From the
Departments of Cell Biology and
Biophysical Chemistry, Hannover Medical School,
30625 Hannover, Germany and ¶Research Group
Molecular Recognition, German Research Centre for Biotechnology, 38124
Brunswick, Germany
Received for publication, April 9, 2003
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ABSTRACT |
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INTRODUCTION |
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According to the current model for the uncoating of clathrin-coated vesicles, it is believed that auxilin first binds to clathrin coats and then recruits hsc70 in an ATP-dependent reaction via its J-domain. The interaction with the J-domain stimulates the ATPase activity of hsc70, thereby stabilizing the binding of hsc70 to clathrin. hsc70 interacts stoichiometrically with the legs of the clathrin triskelia, whereas auxilin functions catalytically in this process. The association of hsc70 with clathrin results in the release of clathrin triskelia by disrupting clathrin-clathrin interactions. Apart from guiding hsc70 to its clathrin substrate, the clathrin-binding domain of auxilin (amino acids 547813) functions as a clathrin assembly protein in vitro when added in stoichiometric amounts to the heavy chain (2).
Like numerous other endocytic proteins, auxilin directly interacts with
both clathrin and AP-2. The clathrin-binding domain contains three DPF
sequences. This motif has previously been shown for other endocytic proteins
to play a role in the interaction with AP-2
(6). However, auxilin lacks
typical clathrin box-binding sequences, which mediate the interaction of a
number of endocytic proteins with the N-terminal domain of clathrin
(clathrin-TD).1 Two
types of clathrin box motifs have been described so far. The clathrin box I
motif follows the consensus sequence L(L/I)(D/E/N)(L/F)(D/E)
(7), whereas the clathrin box
II motif is defined by the motifs WDPW and LMDLA
(8). Instead, two DLL sequences
that have previously been implicated in the clathrin assembly function of
AP180 (9) are present in the
clathrin-binding domain of auxilin. These motifs are tandemly arranged within
this region with the putative AP-2-binding DPF sequences. The affinity for the
ear domain of -adaptin seems to be correlated with the number of
DP(F/W) peptides, which often occur in multiple copies in the sequence of many
accessory proteins (6). An
alternative binding motif for the
-ear domain was recently defined by
the consensus sequence FXDXF
(10). In the case of AP180 and
amphiphysin, the FXDXF motifs seem to contribute to the
binding of the
-ear domain more than the DPF sequences
(11,
12). However, this motif is
not present in auxilin. The
-ear domain of AP-2 consists of a
-sandwich structure and a C-terminal platform domain that are both
engaged in protein-protein interactions. The binding sites of the DPF and
FXDXF peptides overlap on the
-ear platform, with the
latter motif covering an extended site on this subdomain
(13). Despite the low sequence
identity, the respective ear domains of the
- and
-subunits of
AP-2 display a high structural similarity. A subset of DP(F/W)
motif-containing proteins, e.g. AP180, epsin-1 and eps15, binds not
only to the
-ear domain, but also to the
-ear domain of AP-2
(14).
The clathrin heavy chain is organized into a globular N-terminal domain, rod-like distal and proximal domains, and a trimerization region near the C terminus. The N-terminal domain is connected to the distal domain by a flexible linker and can be cleaved off from assembled clathrin by digestion with trypsin without destabilizing the cages. Because auxilin is able to stimulate the hsc70-dependent dissociation of such truncated clathrin cages, it has been suggested that the interaction of auxilin with the proximal or distal domain of clathrin is sufficient for its cofactor activity (1). However, we previously found that auxilin also interacts with clathrin-TD. The clathrin-binding domain of auxilin (amino acids 547813) is bipartite and consists of two subdomains comprising either predominantly clathrin N-terminal or distal domain interaction sites (15). Recently, Ma et al. (16) constructed a chimeric protein from a C-terminal fragment of murine AP180 (residues 325915) and the J-domain of auxilin. With this chimera, they were able to demonstrate that binding of a J-domain protein to the N-terminal domain of the clathrin heavy chain is sufficient to recruit hsc70 and to facilitate the dissociation of clathrin cages to a certain degree.
To characterize the overall structural organization of the clathrin-binding
domain, we employed CD spectroscopy, which showed that this segment of auxilin
is intrinsically disordered. This finding formed the basis for further
dissections of the clathrin-binding domain and for defined mutations within
putative protein-protein interaction motifs. We found that the two short
sequences 636WDW and 674DPF are utilized for the
interaction with the -ear domain of AP-2 and clathrin-TD. Moreover the
674DPF peptide can also interact with the clathrin distal domain.
The 726NWQ sequence was recognized as important for efficient
clathrin assembly by auxilin.
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EXPERIMENTAL PROCEDURES |
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DNA ConstructsAll auxilin constructs used in this study were generated from the DNA of auxilin (Aux)-(547910) cloned into the pGEX-4T2 vector (17). The DNA fragment of Aux-(547715) was amplified by PCR and inserted into the BamHI and SacI sites of pQE-31 (QIAGEN). Cloning of Aux-(715902) into the pET32a vector was described previously in (15). However, for this work we transferred the insert DNA from pET32a into pQE-30 by restriction digestion with NcoI and SalI and ligation between the BamHI and SalI sites of the pQE-30 vector after blunting the NcoI and BamHI overhangs with Klenow polymerase. The construct of Aux-(715902) in pET32a was used to create Aux-(715776) and Aux-(778902). The latter fragment was excised with PvuII and EcoRI and inserted into the EcoRV and EcoRI sites of pET32a, whereas Aux-(715776) was obtained by digestion with NcoI and PvuII and ligated between the NcoI and filled-in EcoRI sites of the pET32a vector. To generate Aux-(715813), the DNA of Aux-(715902) was cleaved with BamHI and introduced into the pQE-30 vector that was linearized with the same enzyme. Aux-(547618) was excised from Aux-(547715) in pET32a using SphI and NcoI and inserted into the NcoI- and EcoRV-cleaved pET32b vector. The resulting SphI overhangs were blunted with mung bean nuclease. The Aux-(619738) fragment was obtained with SphI, subcloned into pUC57 (MBI Fermentas, St. Leon-Roth, Germany), excised with SalI and HindIII, and inserted into the same restriction enzyme sites of the pQE-31 vector. This plasmid was then digested with SalI and StuI, treated with Klenow polymerase, and religated.
Constructs of auxilin mutants were obtained with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) utilizing Aux-(547910), Aux-(547715), Aux-(715902), and Aux-(619738) as templates. Mutagenic primers were used to convert the DPF and DLL sequences into APA and ALA, respectively. 636WDW, 726NWQ, and 753FS were mutated into alanine residues. To generate the auxilin peptide 674DPFADLG, the oligonucleotides 5'-GATCCGATCCTTTCGCTGACCTGGGACGTTAAC and TCGAGTTAACGTCCCAAGGTCAGCGAAAGGATCG-3' were melted at 95 °C for 2 min, annealed at 75 °C for 5 min, and cooled down to 4 °C at a rate of 0.6 °C/min. The double-stranded DNA was ligated with the pET32a expression vector that was linearized with BamHI and SalI. Restriction enzymes and modifying enzymes were obtained from Roche Applied Science (Mannheim, Germany), New England Biolabs Inc. (Frankfurt, Germany), or MBI Fermentas. Verification of the engineered plasmids was performed through DNA sequencing. The point mutation T562A was detected for Aux-(547715) and mutant constructs derived from this fragment.
The recombinant clathrin fragments used in this work and the construct for
His6-AP180-(328896) were described previously
(15,
18). The DNAs for the
glutathione S-transferase (GST) fusion constructs of clathrin-TD and
the -ear domain of AP-2 were kindly provided by Jim Keen (Thomas
Jefferson University, Philadelphia, PA) and Richard Anderson (University of
Texas, Dallas, TX).
Expression and Protein PurificationAll recombinant proteins cloned into the pET32 vector series were transformed into BL21(DE3) bacteria (Merck Biosciences). Protein expression of pQE vector-derived auxilin fragments was performed in M15 bacteria (QIAGEN). The expression and purification protocol was described previously (15). The Aux-(547715) mutant with 579DPF replaced with APA or with 591DLL replaced with ALA was extracted from inclusion bodies according to the recommendations given by QIAGEN. The auxilin fragments Aux-(547715), Aux-(715813), and Aux-(715902) were further purified by ion exchange chromatography in 25 mM Tris-HCl (pH 8.0) using a Mono Q column (Amersham Biosciences, Freiburg, Germany) that retained the contaminations quantitatively while the desired proteins were collected in the flow-through fraction. GST fusion proteins were thrombin (ICN)-digested to remove the GST moiety. hsc70 was prepared from porcine brains according to the protocol for bovine hsc70 (19). Clathrin was extracted from pig brain clathrin-coated vesicles (20) using 0.5 M Tris-HCl (pH 7.0). After centrifugation for 20 min at 266,000 x g in a TLA100.4 rotor (Beckman Coulter Inc., Fullerton, CA), the supernatant was loaded onto a preparative Superose 6 gel filtration column (Amersham Biosciences) equilibrated in 0.5 M Tris-HCl (pH 7.0) at 4 °C. Clathrin fractions without assembly proteins were concentrated overnight by ammonium sulfate precipitation in the presence of 2 mM EDTA (pH 7.0) on ice and pelleted at 72,000 x g for 20 min. To remove any residual ammonium sulfate, the purified clathrin was dialyzed against 0.5 M Tris-HCl (pH 7.0), and assembly into cages was induced by dialysis against buffer A (100 mM MES, 1 mM EDTA, 0.5 mM MgCl2, and 2 mM CaCl2 (pH 6.4)).
Limited Trypsin Digestion of Clathrin CagesTo remove the
N-terminal domains from the clathrin heavy chains, clathrin cages (1.5 mg/ml)
were mildly digested with a final concentration of 0.5 mg/ml trypsin
(Worthington) in a reaction volume of 515 µl in buffer A containing 1
mM CaCl2. After incubation for 15 min on ice and for 50
min at 10 °C, the reaction was terminated with 1.3 mg of soybean trypsin
inhibitor/mg of protease. Trypsin-digested cages were separated by
ultracentrifugation for 20 min at 126,000 x g from the released
N-terminal domains and the trypsin. The cages were washed once with buffer A,
pelleted by ultracentrifugation, and finally resuspended in 90 µl of buffer
B (25 mM HEPES, 12.5 mM potassium acetate, and 5
mM magnesium acetate (pH 7.1)) containing the inhibitor at 0.2
mg/ml.
Limited Trypsin Digestion of Clathrin-coated VesiclesPig
brain clathrin-coated vesicles were mildly digested with trypsin to deplete
endogenous auxilin. 20 µl of clathrin-coated vesicles prepared as described
(20) were incubated with 2
µg/ml trypsin for 30 min on ice in a total volume of 500 µl. To
inactivate the trypsin, 20 µg/ml soybean trypsin inhibitor was added to the
reaction mixture. The vesicles were pelleted by ultracentrifugation, washed
once with phosphate-buffered saline (2.7 mM KCl, 137 mM
NaCl, 1.9 mM KH2PO4, and 8.2 mM
Na2HPO4 (pH 7.0)), and resuspended in 200 µl of
buffer B containing the inhibitor at 5 µg/ml. This treatment quantitatively
inactivated the associated auxilin, whereas many other components of
clathrin-coated vesicles such as AP180 and clathrin heavy and light chains
were not affected. Only the -subunit of AP-2 was sensitive to
degradation, but at least 20% of the starting material remained completely
intact under the digestion conditions used.
Cage Binding1.4 x 1010 mol of the recombinant auxilin fragments were incubated for 30 min on ice with 6.8 x 1011 mol of clathrin assembled into cages in a total volume of 50 µl of buffer B. After centrifugation at 125,000 x g for 30 min, the clathrin pellets were resuspended in 0.5 M Tris-HCl (pH 7.0) in a final volume that corresponded to that of the supernatants. In all subsequent experiments, pellet and supernatant fractions analyzed by SDS-PAGE were directly comparable to each other. Protein bands were quantified from digitalized images using NIH Image Version 1.62 software (National Institutes of Health, Bethesda, MD).
Clathrin AssemblyAssembly experiments were performed as described previously (15).
Uncoating AssayThe cofactor function of recombinant auxilin
fragments was tested in an uncoating reaction that was carried out for 15 min
at 25 °C with auxilin-depleted clathrin-coated vesicles (0.9
µM clathrin); 3.2 µM hsc70; and an
ATP-regenerating system containing 2 mM ATP, 5 mM
creatine phosphate, 1 mM dithiothreitol, and 5 units/ml creatine
kinase in a total volume of 50 µl of buffer B. hsc70 was preincubated for
20 min on ice with the ATP-regenerating mixture and added to the vesicles and
increasing substoichiometric concentrations of auxilin fragments. The released
clathrin was separated from intact cages by ultracentrifugation.
Trypsin-digested clathrin cages were dissociated under the same conditions as
clathrin-coated vesicles using 1.4 µM clathrin and 68
nM recombinant auxilin fragments.
Pull-down ExperimentsBinding and competition experiments
were performed as described
(15) with the following
modifications. GST-ear domain and GST-TD fusion proteins precoupled to
glutathione-Sepharose beads were incubated for1honiceina reaction volume of 50
µl of buffer B with 2 x 1010 mol of the
recombinant auxilin fragments. For competition experiments, the beads were
diluted 1:3 with Sepharose CL-4B, and 1.5 x
1010 mol of the recombinant auxilin fragments
were mixed with an equimolar amount or a 2-, 5-, or 10-fold molar excess of
either His6-AP180-(328896) or the distal domain of
clathrin.
Overlay Assay with an Auxilin Peptide ArrayAuxilin-derived
peptide fragments 15 amino acids in length were chemically synthesized in
situ on a cellulose membrane as described previously
(21,
22). The sequence of each
peptide overlapped with that of the following one by 12 residues spanning
amino acids 570789. In an overlay assay, biotinylated recombinant
proteins of the -ear domain of AP-2 and clathrin-TD were bound to the
auxilin peptide array. Biotinylation was performed with 37.5 µg of EZ-Link
sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce)/mg of protein according to
the manufacturer's protocol. The peptide array was incubated for 1 h at room
temperature with the biotinylated proteins at 1 µg/ml in buffer B
containing 3% bovine serum albumin and washed with ice-cold buffer B. Bound
protein was detected after incubation with horseradish peroxidase-conjugated
streptavidin (Pierce) by chemiluminescence. Signals were visualized on ECL
films (Amersham Biosciences), digitalized, and quantified using NIH Image
Version 1.62 software.
Circular Dichroism SpectroscopyCD spectra were obtained using a Jobin Yvon Dichrograph III with a bandwidth of 2 nm, a scanning rate of 0.03 nm/s, and a time constant of 2 s. All measurements were conducted at room temperature in phosphate-buffered saline with protein concentrations of 0.50.9 mg/ml isolated auxilin fragments, 0.25 mg/ml for experiments with 50% trifluoroethanol, or 1.3 mg/ml of a 1:1 molar ratio of auxilin fragments and clathrin-TD. The spectra were evaluated with CDPro software using the expanded reference set of protein spectra as described previously (23).
Heat DenaturationThe susceptibility of auxilin fragments to irreversible denaturation was tested by heating the proteins in a boiling water bath and shock-cooling them afterward according to the protocol given by Kalthoff et al. (18).
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RESULTS |
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To experimentally address the question about the secondary structure of the
clathrin-binding domain of auxilin, we recombinantly expressed three fragments
and analyzed them by far-UV CD spectroscopy (an overview of all auxilin
fragments used in this study is shown in
Fig. 1). The spectra revealed
that both fragments His6-Aux-(547715) and
His6-Aux-(715813) are almost completely unstructured
(Fig. 2A and
Table I). As expected, only the
His6-Aux-(715902) fragment, which includes the J-domain,
contains a significant amount of -helix. We therefore concluded that
the clathrin-binding region of auxilin is highly flexible. However, there are
numerous examples of intrinsically disordered proteins that gain structure
upon interaction with their binding partners (for an overview, see Ref.
25). For instance, the
isolated clathrin light chains are natively unfolded, but become largely
-helical upon interaction with the clathrin heavy chains
(26). To test whether the
unstructured fragments of auxilin would principally be capable of forming
secondary structure in a suitable environment, we repeated the measurements
with His6-Aux-(547715) and
His6-Aux-(715813) in the presence and absence of
trifluoroethanol, an organic cosolvent known to induce helicity in numerous
peptides and proteins. Upon addition of trifluoroethanol, both fragments
showed an increased content of
-helix, roughly corresponding to the
formation of two short helical stretches with a length of
8 amino acids
(Fig. 2, B and
C). This suggests that at least parts of the disordered
region of auxilin are generally capable of undergoing a conformational change
to
-helices. However, when the fragments were combined in a 1:1 molar
ratio with recombinantly expressed clathrin-TD and analyzed by CD
spectroscopy, no difference was found between the spectra of the mixtures and
the combined spectra of the individual components
(Fig. 2, D and
E). Thus, auxilin apparently does not gain structure upon
interaction with clathrin-TD. Similar observations were previously obtained
for the interaction between the DPW domain of epsin-1 and the
-ear
domain of AP-2 (13). However,
both sets of results need to be viewed with caution because it cannot entirely
be ruled out that, under the conditions used, a low affinity interaction
between the fragments may not form a sufficient amount of complex to generate
a detectable signal. Nevertheless, we think that the chosen protein
concentrations should be high enough to enable an efficient interaction.
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Heat Stability of the Clathrin-binding Domain of Auxilin
The disordered regions of epsin-1 and AP180 are highly soluble and resistant
to irreversible denaturation upon heating to 100 °C
(18). This can be explained by
the lack of a compact fold of the polypeptide chain, even under physiological
conditions. To test whether the clathrin-binding domain of auxilin is also
resistant to heat denaturation, we incubated several recombinantly expressed
fragments of auxilin for 5 min in a boiling water bath, shock-cooled them, and
pelleted precipitated protein by ultracentrifugation. GST was added to the
samples as an internal control. The experiment showed that the unstructured
fragments His6-Aux-(547715) and
His6-Aux-(715813) remained almost quantitatively in the
supernatant. In contrast, the auxilin fragments Aux-(547910) and
His6-Aux-(715902), which contain the J-domain, partly
precipitated, whereas the heat-labile GST coagulated completely and was found
in the pellet fractions (Fig.
3). To examine whether the incubation altered the functional
properties of the heat-stable fragments, we determined the binding
efficiencies of boiled and untreated His6-Aux-(619738) in a
pull-down experiment with the -ear domain of AP-2 and found no
difference (data not shown).
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Identification of Binding Motifs for Clathrin and AP-2 The
clathrin-binding domain of auxilin contains three DPF and two DLL binding
motifs, which have previously been shown to be involved in the interactions of
other proteins with AP-2 and clathrin, respectively. However, it is not known
whether they fulfill a similar function in auxilin. We started with a broad
approach to identify the sequences responsible for the protein interactions of
auxilin by using arrays of auxilin peptides synthesized in situ on
cellulose membranes. Each peptide was a 15-mer that overlapped with the
following one by 12 residues. The arrays covered the segment of amino acids
570789 of auxilin. The membrane strips were probed with recombinantly
expressed clathrin-TD and the -ear domain of AP-2 in overlay assays.
The experiments revealed a surprisingly large number of interactions between
auxilin peptides and the clathrin and AP-2 probes
(Fig. 4). Intriguingly, in most
cases, the interaction sites for clathrin-TD and the
-ear domain of
AP-2 seemed to overlap. Only sequence 591DLL appeared to interact
exclusively with clathrin-TD. All other sequences considered to be involved in
clathrin or AP-2 binding such as the three DPF peptides and the
636WDW sequence, which might be distantly related to clathrin box
II motifs, associated with both clathrin-TD and the
-ear domain of
AP-2. In addition to these interactions, clathrin-TD and the
-ear
domain also bound to peptides showing no similarities to previously described
motifs for clathrin or AP-2 binding. One set of such peptides contains the
short sequence 726NWQ; another one includes the dipeptide
753FS, which is also conserved in auxilin-2. The common denominator
seems to be a large hydrophobic residue such as Trp, Phe, or Leu.
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Analysis of Binding Motifs for Clathrin and AP-2 by Site-directed
MutagenesisTo confirm that the motifs identified by the peptide
arrays are functional in the context of the protein and utilized for the
interaction of auxilin with clathrin and AP-2, we used site-directed
mutagenesis and analyzed the effects in GST pull-down assays and clathrin cage
binding studies. Mutation of motifs 579DPF and 674DPF to
APA and 636WDW to AAA (in combination with motif 674DPF
to APA) all significantly reduced the affinity of auxilin fragments
Aux-(547910), His6-Aux-(547715), and
His6-Aux-(619738) for both GST-TD and GST--ear domain
(Fig. 5, A and
B). Thus, in the context of the auxilin clathrin-binding
domain, these peptides are utilized in dual specific interactions with both
clathrin-TD and the
-ear domain of AP-2. However, it also seems that
the three DPF motifs are not equivalent because mutation of 674DPF
had a much stronger negative effect on binding to the
-ear domain than
the elimination of 579DPF. The double mutation of 605DPF
and 674DPF did not further reduce the affinity for clathrin-TD
compared with the single mutation of 674DPF, whereas a clear effect
on the interaction with the
-ear domain was detectable. Yet, it is
evident that the strength of
-ear domain binding by the auxilin
fragments correlates with the number of DPF motifs in the sequence
(Fig. 5B).
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Converting the two DLL motifs into ALA had the strongest impact on the
binding of auxilin constructs to clathrin-TD
(Fig. 5A). The
association with the -ear domain of AP-2 was not affected, however,
when the DLL peptides were altered (data not shown). Therefore, these
sequences seem to be exclusively responsible for the interaction with
clathrin.
Auxilin fragments failed to be detected by monoclonal antibody 100/4 when the 636WDW sequence was mutated, suggesting that these residues contribute to the epitope for the antibody. Our finding was confirmed by binding of monoclonal antibody 100/4 to the auxilin peptide arrays (data not shown).
Next, we asked whether the identified motifs involved in binding to recombinant clathrin-TD would also play a role in interactions of auxilin with assembled clathrin. We incubated preformed clathrin cages with wild-type and mutant auxilin fragments, pelleted the cages by ultracentrifugation, and analyzed supernatant and pellet fractions by SDS-PAGE and Western blotting for the presence of auxilin fragments. Furthermore, we compared the affinities of the various fragments for intact cages with those from which the N-terminal domains had been removed with trypsin.
First of all, the organization of the clathrin-binding domain into two subdomains became evident by the different affinities of the wild-type constructs His6-Aux-(547715) and His6-Aux-(715902) for clathrin cages. The latter fragment is predominantly involved in binding to the distal or proximal domain of the heavy chain, whereas the former preferentially associates with the N-terminal domain (see also Ref. 15). Yet, His6-Aux-(547715) retained a weak affinity for truncated clathrin cages; and likewise, binding of His6-Aux-(715902) was affected by the removal of the N-terminal domain from cages, implying an interaction with clathrin-TD (Fig. 5C).
Mutations in motifs 579DPF, 674DPF, and
591DLL reduced the association of
His6-Aux-(547715) with assembled clathrin
(Fig. 5C). A further
reduction in binding to the cages was observed when the 636WDW
sequence was eliminated together with the 674DPF motif
(Fig. 5C). An effect
on binding to cages lacking the N-terminal domains was hardly detectable
because the affinity of the wild-type construct was already low
(Fig. 5C). A combined
mutation of the 605DPF and 674DPF sequences did not seem
to further reduce binding of the auxilin fragment to clathrin cages compared
with the single mutation of 674DPF. This reflects the finding that
the 605DPF motif did not play a major role in the interaction with
GST-TD in the pull-down experiments (Fig.
5A). Our mutation analysis singled out the
674DPF sequence as a most versatile protein-protein interaction
module. Its alteration not only affected the binding of auxilin to clathrin-TD
and the -ear domain of AP-2 in pulldown experiments, but also reduced
the interaction of His6-Aux-(619738) with clathrin cages
lacking the N-terminal domains (Fig.
5C). This result indicates that the 674DPF
motif is directly or indirectly involved in the association of
His6-Aux-(619738) with the distal or proximal domain of the
clathrin heavy chain. Thus, the 674DPF motif is engaged in multiple
interactions of auxilin with clathrin and AP-2.
A particularly strong effect on binding of several auxilin fragments to clathrin cages was observed upon mutation of the two DLL peptides within the region of amino acids 547910, especially of the sequence 781DLL (Fig. 5C). This could be seen even with cages from which the N-terminal domains had been removed. Taken together with the results from pull-down experiments with GST-TD, this suggests that both DLL motifs have the potential to bind to the N-terminal domain as well as to the distal or proximal domain of the clathrin heavy chain. The property of binding to truncated cages seemed to be more pronounced for the 781DLL motif.
The auxilin peptide overlay assays revealed sequences 726NWQ and 753FS as putative novel motifs for binding to clathrin-TD (Fig. 4). His6-Aux-(715902), containing both sequences, was divided into the shorter fragments Trx/His6-Aux-(715776) and Trx/His6-Aux-(778902) to investigate the potential binding motifs 726NWQ and 753FS separately from the 781DLL sequence. Both fragments were still able to bind to GST-TD in pull-down experiments, although with different affinities. They also interacted with assembled clathrin irrespective of the presence of the N-terminal domains (Fig. 6). Thus, the region between amino acids 715 and 902 includes more affinity sites for clathrin than just the 781DLL motif located between amino acids 715 and 776. We mutated both the 726NWQ and 753FS sequences in His6-Aux-(715902) and tested their relevance for the interaction with clathrin cages. However, we could detect only a slight reduction in binding of the mutant fragment to truncated cages after elimination of the 753FS sequence (Fig. 5C).
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Overlapping Binding Sites for Clathrin and AP-2The overlay
assays with the peptide arrays and the mutation studies suggested that several
interaction sites like the one including the 674DPF motif are
utilized for binding of auxilin to both clathrin and AP-2. To explore this in
more detail, we used a recombinantly expressed, Trx/His6-tagged
peptide of sequence 674DPFADLG680 in pull-down and cage
binding experiments. We observed that this peptide associated with both GST-TD
and GST--ear domain as well as with intact and trypsin-digested
clathrin cages lacking the N-terminal domain
(Fig. 7A). Thus, the
674DPF sequence was clearly utilized for all of the tested
interactions, which also explains our earlier finding that the
-ear
domain of AP-2 and clathrin-TD compete for binding to the
Trx/His6-Aux-(619738) fragment
(15).
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The principal binding site of auxilin on the clathrin legs had previously
been located in the distal domain of clathrin
(15). Because the
674DPF peptide is involved in interactions with both the
-ear domain of AP-2 and truncated clathrin cages, we predicted that the
-ear and clathrin distal domains might also compete for an interaction
with auxilin. To test this, we incubated His6-Aux-(619738)
with GST-
-ear domain and added increasing amounts of a recombinant
clathrin distal domain fragment (Trx/His6-tagged clathrin distal
domain) to the assay. The experiment clearly demonstrates that the auxilin
fragment was displaced from the
-ear domain of AP-2 by an excess of the
distal domain of clathrin, indicating that both interactions exclude each
other (Fig. 7B).
Role of Clathrin-binding Motifs in the Assembly Activity of AuxilinWhen present in stoichiometric amounts relative to clathrin, auxilin promotes polymerization of clathrin triskelia to cages in vitro (2). To gain insight into the molecular requirements for the assembly activity of auxilin, we examined the effects of the mutant binding motifs on the polymerization of clathrin into cages. We added a 2-fold molar excess of different auxilin fragments and mutant proteins to purified clathrin and separated assembled clathrin cages from free clathrin triskelia by ultracentrifugation. The His6-Aux-(547715) fragment could only poorly assemble clathrin into cages, whereas the C-terminal fragment His6-Aux-(715902) was almost as efficient as Aux-(547910) (Fig. 8A). Knocking out the DLL motifs strongly reduced the ability of the recombinant auxilin fragments to induce clathrin polymerization. It had been shown previously that elimination of the DLL motifs in AP180 also reduces the assembly activity of this protein (9). Mutations of the DPF motifs in auxilin were much less effective. Unexpectedly, however, the substitution of 726NWQ with AAA resulted in a reduction of the assembly activity of His6-Aux-(715902) by >40%, whereas mutation of 753FS had only a slight effect. Therefore, the most important sequences involved in clathrin assembly seem to be 726NWQ and 781DLL.
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However, the existence of only one of the latter motifs in a fragment proved insufficient to promote clathrin polymerization because neither of the fragments Trx/His6-Aux-(715776) and Trx/His6-Aux-(778902) was effective in an assembly experiment, although both fragments were still capable of interacting with GST-TD and trypsin-digested cages (Figs. 6 and 8B).
Similarities between the Clathrin-binding Domains of AP180 and
AuxilinBoth auxilin and AP180 interact with clathrin-TD as well as
with the -ear domain of AP-2 and are able to support the polymerization
of clathrin to cages. Many of the short peptide sequences that we have
identified for these interactions in auxilin are also present in AP180. We
already showed that auxilin and AP180 compete for clathrin-TD
(15). Therefore, we asked
whether both proteins bind to the same sites on AP-2. This was tested by
incubating His6-Aux-(547715) with GST-
-ear domain in
the presence of increasing amounts of His6-AP180-(328896). A
2-fold molar excess of AP180 displaced the auxilin fragment by more than half.
Thus, we conclude that AP180 and auxilin also compete for binding to AP-2,
indicating that they interact with the same site on the
-ear domain
(Fig. 9A).
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It was previously demonstrated that AP180 can associate via its DPF motifs
not only with the -ear domain, but also with the
2-ear
domain of AP-2 (14). This
raised the question of whether the same would be true for auxilin. Therefore,
we performed a GST pull-down experiment and showed that the
2-ear domain bound also directly to auxilin
(Fig. 9B).
Role of Interactions with Clathrin and AP-2 in the Uncoating Activity
of AuxilinBecause we observed strong effects on the interaction of
auxilin with clathrin and AP-2 after mutating putative binding motifs, it was
of interest to see how the elimination of these motifs influenced the ability
of the auxilin fragments to stimulate the uncoating of clathrin-coated
vesicles. To address this question, we used clathrin-coated vesicles mildly
treated with trypsin to inactivate endogenous auxilin. Although clathrin was
unaffected under the chosen conditions, we could ensure that a significant
amount of -adaptin was not degraded by the protease. The
trypsin-treated coated vesicles were incubated with hsc70, an ATP-regenerating
system, and wild-type or mutant fragments of auxilin. Intact coated vesicles
were separated from released clathrin by ultracentrifugation.
Mutations of the 674DPF sequence in Aux-(547910) as well as the 726NWQ and 753FS sequences in His6-Aux-(715902) had only a minor but detectable effect on the release of clathrin from the vesicle coats. However, the loss of motifs 591DLL and 781DLL led to a dramatic reduction in the uncoating activity of the fragments (Fig. 10, A and B). Therefore, both DLL sequences of Aux-(547910) play important roles in the hsc70-mediated dissociation of the clathrin coat. Interestingly, the N-terminal truncation of His6-Aux-(715902), which quite efficiently functions in the coat disassembly, to Trx/His6-Aux-(778902) resulted in an almost complete loss of uncoating activity (Fig. 10C). Therefore, the J-domain and the 781DLL motif are essential, but not sufficient, for coat dissociation induced by Aux-(715902) in the presence of hsc70. The segment between amino acids 715 and 778 seems to make an important contribution to the cofactor function of auxilin in this assay.
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Clathrin cages with the N-terminal domains removed by trypsin digestion can be disassembled in vitro by hsc70 in the presence of auxilin (1). We tested whether the mutations in Aux-(547910) would affect the disassembly of such cages. Again, alterations in the DLL motifs resulted in a strong reduction of the ability of the auxilin fragment to mediate the release of clathrin triskelia from the cages, whereas mutation of 674DPF had only a small effect (Fig. 10D). This result confirms that both DLL sequences in Aux-(547910) are essential for a productive interaction of auxilin with the proximal and distal domains of clathrin and its cofactor activity in coat dissociation.
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DISCUSSION |
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We investigated the structural organization of the clathrin- and
AP-2-binding domain of auxilin by CD spectroscopy and observed that this
region is almost completely disordered and has a high content of random-coil
conformation. Similar structural properties were previously reported for the
clathrin- and AP-2-binding segments of epsin-1 and AP180
(13,
18). Although auxilin has an
increased helical content in solvents favorable to helix formation, we were
not able to observe that this structure is induced upon incubation with
clathrin-TD. Thus, it seems that the central region of auxilin remains overall
disordered, even when associated with its interaction partners. All of the
identified binding sequences are tandemly arranged within this domain and
consist of short linear peptides. A flexible clathrin-binding domain could
enable auxilin to engage several interaction partners simultaneously, thus
allowing for cooperative binding to clathrin and AP-2. Many endocytic proteins
contain multiple copies of the DP(F/W) sequence, and the strength of the
interaction with AP-2 seems to correlate with their number
(6). Using mutant fragments of
auxilin with successively eliminated DPF motifs, we could now show that these
motifs bind cooperatively to the -ear domain of AP-2. The observation
that the clathrin-binding domain is intrinsically disordered and contains
multiple, alternating short interaction motifs also explains why it was
possible to divide the clathrin-binding domain into several shorter fragments
that retained clathrin- and AP-2-binding activities.
We also identified novel binding sequences for clathrin and AP-2 that do not seem to be related to previously described motifs. These are sequences 636WDW, 726NWQ, and 753FS. Moreover, we noted that the 579DPF, 674DPF, and 636WDW motifs were able to engage both AP-2 and clathrin and thus represent binding sequences with dual specificity (an overview of the identified binding determinants and their interaction partners is given in Fig. 11). Similar binding characteristics were previously assigned to the PWDLW binding sequence in human amphiphysin-2 (8).
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To what extent interactions with binding sequences of multiple
specificities can impede each other became apparent from our competition
studies. As we had previously shown for the N-terminal domain
(15), the distal domain of the
clathrin heavy chain was able to interfere with the binding of
His6-Aux-(619738) to the -ear domain of AP-2.
Furthermore, the
-ear domain of AP-2 was also able to displace
His6-Aux-(547715) from
clathrin-TD.2 The
design of a clathrin-binding domain with multiple low affinity monospecific
and dual specific interaction sequences arranged along a flexible polypeptide
chain and with a high content of serine and threonine residues would be
perfectly suited for regulation by phosphorylation. Computational analysis of
the auxilin sequence using the PROSITE Database
(27) predicts, for instance,
sequence 588SGQD, which overlaps with the 591DLL
peptide, to be a potential substrate for casein kinase-2. This enzyme has
previously been shown to be present in the protein coats of clathrin-coated
vesicles (28,
29). Such phosphorylation
sites within or in close proximity to binding determinants could be utilized
to selectively raise or lower the affinity of auxilin for one or the other
interaction partner. However, so far, we do not know whether auxilin is a
target for kinases.
Our study has shown that the interaction of auxilin with clathrin is not mediated by binding sequences of the canonical clathrin box I and II types that have been implicated in the association with clathrin-TD. Instead, auxilin utilizes clathrin-binding determinants that include the above-mentioned sequences 636WDW, 579DPF, and 674DPF. Furthermore, the clathrin-binding domain contains two repeats of the DLL sequence that are involved in the interactions with clathrin. For AP180, this motif was implicated in the assembly of clathrin, presumably by cross-linking the N-terminal domains of three adjacent triskelia (9). In fact, auxilin was originally also identified as a clathrin assembly protein (2) before its role in the uncoating of clathrin-coated vesicles was recognized. Our data confirm the role of the DLL sequences in the assembly function of auxilin, but they also show that the assembly mechanism involves not only their association with the N-terminal domains, but also binding to the distal domains of the clathrin heavy chain. This conclusion is based on our previous finding (15) that the clathrin-binding domain of auxilin can be further divided into two functionally distinct subdomains represented by the two auxilin fragments His6-Aux-(547715) and His6-Aux-(715902). The C-terminal auxilin fragment, which is most efficient in clathrin assembly, seems to interact preferentially with the distal domains of the clathrin heavy chain and associates only poorly with the globular N-terminal domain. In contrast, His6-Aux-(547715), which only poorly supports assembly, displays complementary binding specificities with a preference for clathrin-TD (15). Therefore, we propose that the interaction of auxilin with the distal domain of clathrin is essential for driving efficient clathrin assembly.
Direct evidence for involvement of the DLL peptides in binding to the N-terminal and distal domains of clathrin was obtained from mutation analysis. The ability of auxilin to polymerize clathrin was reduced upon substitution of the DLL sequences with ALA. Furthermore, the elimination of these motifs resulted in impaired binding to trypsin-digested cages lacking the N-terminal domains and the clathrin light chains. However, our data also indicate that the presence of one DLL motif is not sufficient for assembly function because Trx/His6-Aux-(778902), which contains the 781DLL sequence, displayed no clathrin assembly activity. In contrast, His6-Aux-(715902) efficiently promoted clathrin assembly. This fragment contains the 726NWQ peptide, which was considered a candidate for a novel clathrin-binding motif on the basis of the peptide array overlay assay. We demonstrated that the 726NWQ sequence is involved in the assembly activity of auxilin. Its elimination from His6-Aux-(715902) significantly reduced the ability of this fragment to promote clathrin polymerization, but did not abolish it completely. Taken together, the 726NWQ sequence synergistically supports the 781DLL peptide in clathrin assembly. Evidence for a role of the 726NWQ sequence in this function is also indirectly provided by the failure of an auxilin fragment ranging from amino acids 728 to 910 to drive clathrin assembly (16).
Quantitative clathrin assembly studies using auxilin fragments with systematically mutated putative binding sequences revealed a small, but significant effect when the 674DPFADL sequence was changed to APAADL. A related sequence (885DPLADL) occurs together with 787NWQ in a C-terminal fragment of AP180 (C16) that was shown by Morgan et al. (9) to process moderate clathrin assembly activity, although it lacks DLL motifs. Thus, it seems that different endocytic proteins utilize similar combinations of identical or related short binding sequences to promote clathrin polymerization.
Taking into account the number of clathrin interaction sites within the
clathrin-binding domain of auxilin, a correct spacing seems to be a further
requirement for efficient binding and assembly activity. This could explain
why binding motifs for clathrin-TD within His6-Aux-(547715)
can only poorly assemble clathrin, whereas they might be important in the
context of the entire clathrin-binding domain of Aux-(547910). This
idea is supported by the clathrin-binding studies with deletion mutants of the
-hinge domain of AP-1 and of amphiphysin-2. Decreased distances between
interaction motifs result in a partial or complete loss of clathrin-binding
properties (8,
30).
It was recently reported that Hip1 (Huntingtin interacting protein-1)
directly links coat components to the actin cytoskeleton. It associates with
clathrin light chains via a central helical domain that contains a DLL motif.
Although Hip1 does not interact with the isolated heavy chains, it still can
drive clathrin assembly (31,
32). Moreover, preliminary
reports show that mutation of this DLL motif abolishes its assembly activity
(33). So far, we could not
detect an association of GST-Aux-(547910) with purified clathrin light
chains in pull-down experiments (data not shown), but this result does not
rule out an interaction of auxilin with light chains once they are bound to
the heavy chain. Chen et al.
(26) have shown that the light
chains gain -helical structure upon binding to the heavy chains. This
raises the possibility that an appropriate binding pocket for auxilin might
not form unless clathrin light and heavy chains are associated. We do not know
yet whether any of the auxilin DLL sequences recognize the light chains on
clathrin triskelia. A negative regulatory function of the light chains in
clathrin assembly is widely accepted
(34,
35); and therefore, a role of
assembly proteins that result in a conformational change of the light chains
is an attractive hypothesis.
The physiological significance of the clathrin assembly function of auxilin is not yet clear. It is possible that the assembly activity of auxilin has functional relevance at early stages of the recruitment of coat components to the membrane. However, we favor the explanation that the assembly-promoting activity of auxilin reflects its much higher affinity for polymerized clathrin than for free triskelia. It is thereby ensured that hsc70 is efficiently placed on clathrin coats only. Consequently, stoichiometric amounts of auxilin relative to clathrin would shift the equilibrium from free triskelia to assembled clathrin.
From this and previous work (1), we know that auxilin is able to mediate the dissociation of clathrin cages that lack the N-terminal domains. This raises the obvious question of why auxilin interacts with clathrin-TD. Ma et al. (16) recently showed that a chimera of the clathrin-binding region of AP180, which binds exclusively to clathrin-TD, and the J-domain of auxilin is able to mediate the dissociation of clathrin cages to a certain extent. However, this construct was far less efficient than auxilin. Thus, for proper binding to clathrin and efficient placement of hsc70 on the clathrin legs, interaction with both the N-terminal and distal domains seems to be required. We identified several motifs, including both DLL peptides, as well as the 674DPF and 753FS sequences to be involved in binding to the distal domain. Mutation analysis revealed that converting these motifs into inactive residues resulted in an impaired uncoating activity. The most dramatic effect was seen upon substitution of the DLL motifs with ALA.
Based on our data, we present the following model for the function of
auxilin in the uncoating reaction. We propose that auxilin initially binds to
the clathrin N-terminal domains underneath the vertex of the triskelion to be
released from the cage (triskelion A)
(Fig. 12). These N-terminal
domains belong not to the direct neighbors, but to the ones further away in
the lattice. Then, auxilin interacts via its C-terminal clathrin-binding
domain with the distal domain of an adjacent triskelion to direct an hsc70
molecule to triskelion A. Auxilin functions catalytically by remaining bound
to clathrin-TD via the N-terminal region of its clathrin-binding domain until
all hsc70 molecules are delivered to the proximal domains of triskelion A.
This implies that single clathrin triskelia are released from the cage. Our
model also reflects the functional subdivision of the clathrin-binding domain
of auxilin. However, the observed association with AP-2 may be of importance
for the recruitment of auxilin and the regulation of the uncoating process. It
might also be possible that this interaction provides an alternative binding
site to clathrin-TD because both the -ear domain of AP-2 and the
N-terminal domain of the clathrin heavy chain are in close proximity within
the protein coat. Therefore, the association of auxilin with AP-2 could also
be functionally relevant in the uncoating of clathrin-coated vesicles.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Cell Biology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-6744; Fax: 49-511-532-3903; E-mail: ungewickell.ernst{at}mh-hannover.de.
1 The abbreviations used are: clathrin-TD, clathrin N-terminal domain; Trx,
thioredoxin; Aux, auxilin; GST, glutathione S-transferase; MES,
2-(N-morpholino)ethanesulfonic acid.
2 U. Scheele and E. Ungewickell, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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