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Clathrin- and AP-2–binding sites in HIP1 uncover a general
assembly role for endocytic accessory proteins.
Sanjay K. Mishra*, Nicole R. Agostinelli*, Tom J. Brett¶, Ikuko Mizukami†,
Theodora S. Ross† and Linton M. Traub*§.
*Department of Cell Biology and Physiology, University of Pittsburgh School of
Medicine, Pittsburgh, PA 15261
¶Department of Pathology, Washington University School of Medicine, St. Louis
MO 63110
†Department of Internal Medicine, University of Michigan Comprehensive
Cancer Center, Ann Arbor, MI 48109
§ To whom correspondence should be addressed at:
Department of Cell Biology and Physiology
University of Pittsburgh School of Medicine
3500 Terrace Street, S325BST
Pittsburgh, PA 15261
Tel: (412) 648-9711
Fax: (412) 648-9095
e-mail: traub+@pitt.edu
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on September 27, 2001 as Manuscript M108177200 by guest on A
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Running title: HIP1 binds core endocytic components
Abbreviations
BSA, bovine serum albumin
ENTH, epsin N-terminal homology
GSH, glutathione
GST, glutathione S-transferase
HC, heavy chain
HIP1, huntingtin-interacting protein 1
HIP1R, huntingtin-interacting protein 1 related protein
LC, light chain
PtdIns(4,5)P2, phosphatidylinositol(4,5) bisphosphate
SDS-PAGE, SDS-polyacrylamide gel electrophoresis
WT, wild type
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Clathrin-mediated endocytosis is a major pathway for the internalization of
macromolecules into the cytoplasm of eukaryotic cells. The principle coat
components, clathrin and the AP-2 adaptor complex, assemble a polyhedral
lattice at plasma membrane bud sites with the aid of several endocytic accessory
proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding
partner of huntingtin, co-purifies with brain clathrin-coated vesicles and
associates directly with both AP-2 and clathrin. The discrete interaction
sequences within HIP1 that facilitate binding are analogous to motifs present in
other accessory proteins, including AP180, amphiphysin and epsin. Bound to a
phosphoinositide-containing membrane surface via an ENTH domain, HIP1
associates with AP-2 to provide coincident clathrin-binding sites that together
efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis
and the similar modular architecture and function of HIP1, epsin and AP180
suggest a common role in lipid-regulated clathrin lattice biogenesis.
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Endocytosis entails the preferential recruitment of select molecules into a patch
of plasma membrane that will bud into the cytoplasm. At the bud site, an
assembling clathrin coat links the mechanical process of invagination to cargo
selection. The core endocytic components, clathrin trimers and the AP-2
heterotetramer, as well as lipids (1) and multiple protein cofactors, collectively
termed endocytic accessory proteins (2), participate in bud nucleation, lattice
assembly and invagination, and in the final scission event (1-3). The precise role
of many of the accessory proteins remains poorly understood however. One
common feature of several of the accessory proteins is the capacity to bind to
both AP-2 and clathrin (4-13). Associations with the AP-2 adaptor complex
generally involve the independently folded appendage domains of the large α
(αA or αC isoform) and β2 subunits, each separated from the heterotetrameric
adaptor core by a flexible hinge. Despite only ~10% sequence identity, the fold of
the β2 appendage (14) is structurally analogous to that of the α appendage
(15,16), and, indeed, the αC and β2 appendages interact with an overlapping
group of partner proteins (14).
The privileged association of accessory factors with the core endocytic machinery
is due to discrete interaction sequences located within each accessory protein. In
epsin and eps15, the AP-2 αC-binding sequence is Asp-Pro-Trp (DPW) and Asp-
Pro-Phe (DPF) respectively, each protein bearing multiple triplets arrayed in a
tandem fashion. The minimal region of amphiphysin I (12) or AP180 (8) required
to bind the αC appendage does not contain a DPF/W sequence however. Instead,
in these proteins, and the long-splice isoform of phosphoinositide
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polyphosphatase synaptojanin, SJ170, an alternate binding motif is based upon
the di-aromatic consensus sequence FXDXF (where X is any amino acid)1.
Several of the designated AP-2–binding accessory proteins also interact directly
with clathrin. The most prevalent binding motif that facilitates clathrin
association is based upon the consensus L[L,I][D,E,N][L,F][D,E] (6,17,18), the so-
called clathrin box (19). Variations on this type I consensus are found adjacent to
the AP-2 binding sequences in amphiphysin (6,12), epsin (7,9,10), AP180 (13,20)
and β-arrestin (21). The extended type I clathrin-binding sequence interacts with
an elongated shallow cleft in the globular amino-terminal domain of the clathrin
heavy chain (19). Here, we identify an additional protein, huntingtin-interacting
protein 1 (HIP1), which displays canonical AP-2- and clathrin-binding motifs as
well as overall domain organization and functional properties akin to epsin,
AP180 and amphiphysin. Our data strongly implicate HIP1 in clathrin-mediated
endocytosis.
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EXPERIMENTAL PROCEDURES
Construct preparation—Glutathione S-transferase (GST)-HIP1M1 (residues
311–394) and M2 (residues 311–369) were prepared using appropriate PCR
primers and human EST xd79f07.x1 (Research Genetics) as the template. The
inserts were ligated, after digestion, into EcoRI/XhoI-cleaved pGEX-4T-1. A
similar PCR-based strategy was used to insert the human HIP1M1 segment into
pcDNA3.1 with an amino-terminal myc-epitope tag. The GST-mHIP1 (1-533)
fusion construct was prepared to contain in-frame insertion of nucleotide 1–1599
of mouse HIP1. GST-SJ170C2 (residues 1454–1530) and GST-epsin 1 (1–407) were
prepared similarly using human EST nf51b08.s1 (Incyte Genomics) and rat epsin
1 cDNA, kindly provided by Pietro De Camilli, respectively. Mutations were
generated using appropriate mutagenic primers with the QuikChange kit
(Stratagene) as described elsewhere (10,16). All constructs and mutations were
confirmed by automated dideoxynucleotide sequencing.
Cytosol and Protein purification—Cytosol was prepared from frozen rat brains
exactly as described previously (22). The rat brain detergent extract was prepared
as described elsewhere (12) and filtered into assay buffer (25 mM Hepes-KOH,
pH 7.2, 125 mM potassium acetate, 5 mM magnesium acetate, 2 mM EDTA, 2
mM EGTA, 1 mM DTT) containing 0.5% Triton X-100 before use. GST-fusion
proteins were produced in E. coli using a standard IPTG induction protocol
(10,16). After lysis in B-PER (Pierce), soluble protein was purified on glutathione
(GSH) Sepharose, eluted with 25 mM Tris-HCl, pH 8.0, 10 mM GSH, 5 mM DTT
and then dialyzed into PBS, 1 mM DTT. Human HIP1M1 (residues 311-369),
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HIP1M2 (residues 311–369) and epsin DPW (residues 249-407) were cleaved
from GST with thrombin, followed by addition of the irreversible thrombin
inhibitor PPACK (Calbiochem) to a final concentration of 25 µM. For the GST-
mHIP1 (1-533), the purified recombinant protein was first dialyzed to remove
GSH and then treated with thrombin to cleave off the HIP1 segment. Liberated
GST was removed by adding a second aliquot of GSH-Sepharose and collecting
the unbound fraction as mHIP1 (1-533). Clathrin and AP-2 were purified from rat
brain clathrin-coated vesicles by Tris extraction followed by sequential
chromatography over Superose 6 and hydroxylapatite (23). Pooled clathrin- or
AP-2-containing fractions were gel-filtered into assay buffer and centrifuged at
134,000 Xgmax before use in binding assays.
Binding assays—For pull-down-type assays, GST or GST- fusion proteins were
first immobilized on GSH Sepharose, washed in assay buffer and then mixed
with clarified rat brain cytosol/detergent extract to give a final concentration of
~7.5 mg/ml in 300 µl total volume (10,24). After incubation at 4°C for 60 min, the
beads were separated by centrifugation and aliquots corresponding to 1/80 of
each supernatant and 1/8 of each washed pellet were analyzed by SDS-PAGE
and immunoblotting exactly as described (10,16,24).
The two-stage liposome binding assays used synthetic liposomes composed of
10% (w/w) cholesterol, 30% (w/w) phosphatidylethanolamine, 30% (w/w)
phosphatidylcholine and 30% (w/w) phosphoinositides (Sigma) prepared as
described elsewhere (25). Aliquots of 10 µg of mHIP1 (1-533), GST-epsin (1-407),
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AP-2, or combinations thereof, were first preincubated with 0.5 mg/ml
liposomes for 60 min at 4°C. After centrifugation at 20,000 Xgmax for 15 min, the
lipid pellets were resuspended in assay buffer, then 5 µg clathrin added and the
volume adjusted to 200 µl. Both stages contained 0.1 mg/ml BSA as a protein
carrier. After a further 60 min incubation at 4°C, the liposomes were again
centrifuged and the pellets resuspended in SDS-sample buffer. Aliquots
corresponding to 1/25 of each supernatant and 1/4 of each pellet were analyzed
by SDS-PAGE.
Transient transfections—COS-7 cells were grown at 37°C in DMEM supplemented
with 10% fetal calf serum and 2 mM L-glutamine. Cells plated on poly-L-lysine-
coated round glass coverslips were transfected with DEAE dextran and, after 48
hours, incubated in serum-free DMEM for 60 min. Biotinylated human
transferrin (25 µg/ml) was then added and incubation continued for 15 min at
37°C. Cells were fixed with 3.7% formaldehyde and processed for
immunofluorescence microscopy as described (10).
Antibodies—Polyclonal antibodies directed against HIP1 were generated by
immunizing rabbits with the GST-HIP1M1 fusion protein. Anti-HIP1 antibodies
were affinity purified on thrombin-cleaved HIP1M1 coupled to CNBr-activated
Sepharose 4B using standard procedures. The sources of the antibodies against
clathrin, the AP-2 subunits, epsin, amphiphysin, AP180 and eps15 have been
described (10,16,24). Anti-myc mAb 9E10 was from BAbCo and the anti-
synaptotagmin I mAb was purchased from Transduction Laboratories.
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RESULTS
Searching the databases for additional proteins that contain the AP-2 binding
FXDXF motif we identified HIP1. This protein was initially discovered in screens
for binding partners of huntingtin (26,27), the product of the gene that undergoes
pathogenic CAG-codon expansion in Huntington’s disease. HIP1 displays a
modular domain architecture: an epsin N-terminal homology (ENTH) domain
precedes a central region predicted to have a high propensity for coiled-coil
formation, followed by a C-terminal talin-homology I/LWEQ domain (Fig. 1A).
Within the central region of the protein, before the coiled-coil segment, a stretch
of three interwoven FX[N/D/S]X[F/L] motifs proceeds a single DPF triplet (Fig.
1B). Significantly, this region of HIP1 is quite divergent from HIP1R (Fig. 1B), a
related protein roughly 50% identical to HIP1 that localizes to clathrin-coated
pits (28). The presence of these motifs within HIP1 is intriguing as the
intracellular staining pattern of HIP1 is highly reminiscent of AP-2 and clathrin
(29), and the Saccharomyces cerevisiae HIP1/HIP1R orthologue, Sla2p/End4p, is
implicated in endocytic control (30,31). In fact, we find that in rat brain extracts,
HIP1 copurifies with clathrin-coated vesicles (Fig. 2, lane d). Affinity purified
anti-HIP1 antibodies detect a major ~120-kDa polypeptide with a distribution
that, on subcellular fractionation, clearly parallels that of both clathrin and the
AP-2 adaptor complex (Fig. 2). Like HIP1, a major fraction of AP180 is also
recovered in the clathrin-coated vesicle fraction also containing the recycling
synaptic vesicle transmembrane protein synaptotagmin I (right panel, lane d)
(5,23). By contrast, although present, other accessory proteins, including epsin 1
and amphiphysin are not correspondingly enriched within the purified coated
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vesicle fraction (lane c compared to lane d) and substantial cytosolic pools of
these proteins exist (lane c) (5). These results show that HIP1 is significantly
enriched in clathrin-coated endocytic vesicles found in brain.
HIP1 associates directly with the AP-2 adaptor—To assess whether the region
harboring the FXDXF/DPF-type sequences does actually facilitate an interaction
with AP-2, brain cytosol was incubated with GST-fusion proteins containing this
region of human HIP1. The HIP1M1- and M2-fusion proteins (see Fig. 1A) both
pull down AP-2 near quantitatively (Fig. 3A, lane f and h), whereas GST alone
fails to interact with the adaptor complex (lane b), which remains in the
supernatant (lane a). The presence of the µ2 adaptor subunit verifies that
heterotetrameric AP-2 adaptor complexes bind and, in these assays, the extent of
AP-2 association with the HIP1 fusions is similar to that seen with the GST-
SJ170C2 fusion (lane d), which contains residues 1454-1530 of human SJ170
harboring the sequence 1462GFKDSF.
Importantly, the soluble, native HIP1 protein also binds to the isolated αC-
appendage domain of AP-2. When incubated with brain cytosol, GST-αC binds to
AP180, epsin, (Fig. 3B, lane f), eps15, and amphiphysin I and II (see Fig. 3C)
(15,16) but, as there is very little HIP1 in cytosolic extracts (Fig. 2), only low level
HIP1 association is observed (Fig. 3B, right panel, lane f). HIP1 is more abundant
in a rat brain detergent extract (12) (Fig. 3B, lane c) and, after incubation with this
extract, a prominent additional ~120-kDa band is recovered together with the
common binding partners on the GST-αC beads (Fig. 2B, left panel, lane h
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compared to lane f). This major polypeptide is confirmed to be HIP1 by
immunoblotting (right panel, lane h). The substantially enhanced recovery of
native HIP1 on the GST-αC beads without a concomitant increase in the other
appendage partners again argues that the HIP1–AP-2 interaction is direct.
To confirm that HIP1 engages the same binding surface on the αC appendage
utilized by other endocytic accessory proteins, we tested the capacity of the AP-
2-binding region of HIP1 to inhibit the association of other cytosolic accessory
proteins with the immobilized GST-αC appendage. As a control, supplementing
brain cytosol with 20 µM epsin 1 DPW domain (which contains eight tandemly
arrayed DPW triplets) completely abrogates amphiphysin and AP180 binding
and only low level epsin and eps15 association remains (Fig. 3C, lane f) (15).
Since the added DPW domain abolishes associations when bound
substoichiometrically to the αC appendage (left panel, lane f), a single DPW
domain is capable of engaging multiple appendages simultaneously. By contrast,
addition of 20 µM HIP1M2 affects neither epsin nor eps15 binding (lane h).
Instead, this concentration of the HIP1 fragment totally inhibits AP180 binding
and partially interferes with the amphiphysin interaction (lane h). Thus, the HIP1
fragment inhibits the αC partner associations in the reverse order of their
apparent affinity for GST-αC (epsin ≈ eps15 > amphiphysin > AP180)(16); the
cooperative effect of the multiple DPF/W triplets in eps15 and epsin resists
efficient HIP1 competition under these conditions2.
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DPF and FXDXF sequences both bind directly to the αC appendage1. To gain
some insight into the relative contribution of these two sequences within HIP1 to
AP-2 binding, the DPF triplet was altered to KPS using site-directed
mutagenesis. This substitution severely compromises the ability of the GST-
HIP1M2 fusion to associate with soluble AP-2 (Fig. 3A, lane i and j) but clearly
does not abolish adaptor binding totally. We conclude from these experiments
that HIP1 binds to AP-2 directly by engaging the common binding surface on the
platform subdomain of the αC appendage and that the distal DPF sequence
appears to be the dominant ligand. We attribute the remaining adaptor binding
observed with the DPF mutant to the proximal FXDXF motif. Significantly, all of
the known accessory proteins containing the FXDXF motif (amphiphysin, AP180,
SJ170, HIP1) have one or more DXF/W triplets located adjacent to this sequence.
HIP1 also binds to clathrin trimers directly— In addition to binding to AP-2
adaptors, the HIP1–GST-fusion proteins also affinity purify a prominent ~180-
kDa polypeptide (Fig. 3A, left panel, lane f and h). Antibodies identify this
protein as the clathrin heavy chain and the cognate light chains, although not
visible on the stained gel, are also detected on the blots3. It is well established
that the hinge and appendage regions of the AP-2 β2-subunit interact with
clathrin directly (14,32), yet the observed association of GST-HIP1 with clathrin is
not simply due to the presence of bound AP-2. No clathrin associates with the
GST-SJ170C2 fusion protein, despite it binding similar amounts of AP-2 (lane d).
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Closer inspection of the HIP1 sequence reveals a putative clathrin box proximal
to the AP-2-binding elements (Fig. 1B). The sequence, LMDMD, is an unusual
variation on the type I consensus L[L,I][D,E,N][L,F][D,E] (32) which binds to a
shallow groove located between blades 1 and 2 of the clathrin heavy chain β-
propeller (19). The role of this region in clathrin binding was probed by altering
the sequence to AAAMD (LMD→AAA; Fig. 4). Compared to the native sequence
(lane d), this mutation abolishes clathrin binding completely but leaves the AP-2
interaction intact (lane f). This result implicates the LMDMD in binding clathrin
directly but, interestingly, in the GST-HIP1M2 (DPF→KPS) mutant, which still
contains an intact clathrin box, clathrin binding is strongly diminished (Fig. 3A,
lane j). These results are identical to the behavior of the clathrin-binding
sequence 257LMDLADV located within the DPW domain of rat epsin 1, proximal
to the tandemly arrayed eight DPW repeats (10). There, again, robust clathrin
binding is dependent upon AP-2 recruitment, providing coincident binding
motifs for the terminal domain of the clathrin heavy chain. Transient transfection
of a myc-tagged HIP1M1 segment (residues 311-394) into COS-7 cells, like the
AP-2/clathrin-binding DPW domain of epsin 1 (5), inhibits endocytic transferrin
uptake (Fig. 5), although complete inhibition is only seen in less than a third of
all transfected cells.
HIP1 as an endocytic accessory protein—HIP1 also resembles endocytic accessory
proteins in other respects (Fig. 1A). Like amphiphysin and epsin, there is a
unique functional module at the carboxyl terminus; in HIP1 the talin-like
I/LWEQ domain. And like epsin and AP180, sequence analysis algorithms
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predict HIP1 to have ENTH domain. Importantly, the critical Lys and His
residues in AP180 required to electrostatically coordinate the negatively charged
phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) headgroup (13) are
conserved in both HIP1 and HIP1R (Fig. 6A) and indeed murine HIP1 (1-533)
binds to polyphosphoinositides (see below).
To test the hypothesis that one general function of accessory proteins with this
type of domain architecture is to cooperate with AP-2 to drive clathrin lattice
assembly, we assayed clathrin recruitment onto synthetic liposomes containing
PtdIns(4,5)P2. The ENTH domain allows the amino-terminal segments of mouse
HIP1 (residues 1-533) (Fig. 6B, lane d) and epsin 1 (33) (GST-epsin 1 (1-407), lane
j) each to bind directly to phosphoinositide-containing liposomes. AP-2 (lane f)
also associates with the liposomes directly (13), due to a PtdIns(4,5)P2-binding
determinant found at the amino terminus of the α subunit (34). Attached to the
liposome surface, each of these proteins is able to interact with and recruit
soluble clathrin (lane d, f and j), while no clathrin sediments with the liposomes
in their absence (lane b). Mixing either the HIP1 or epsin protein together with
AP-2 increases the recovery of the adaptor complex with the liposome (lane h
and l), validating the interaction surfaces mapped using the affinity interaction
assays with GST (Fig. 3). Importantly, soluble clathrin recruitment onto the
liposome surface is markedly more efficient in the presence of both AP-2 and an
AP-2– and clathrin-binding accessory protein (HIP1, lane h and epsin 1, lane l
compared to lanes d, f and j). These results resemble very closely data obtained
recently using AP180 and AP-2 to assemble clathrin lattices upon synthetic lipid
membranes (13). Thus, several membrane-bound accessory proteins can similarly
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cooperate with AP-2 to effect efficient clathrin assembly. The close similarity in
the behavior of HIP1 and epsin/AP180 in these assays clearly includes HIP1
within the family of endocytic accessory proteins.
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DISCUSSION
Huntington disease is a neurodegenerative disorder principally affecting striatial
neurons, yet the mutated gene product, huntingtin, is not a brain-restricted
protein. Both mRNA and protein is widely expressed in mammalian tissues
(35,36). Broad tissue distribution is true also of both HIP1 and HIP1R (28,29),
hinting at a more general function for these proteins. The intracellular
distribution of huntingtin overlaps partly with clathrin (37,38) and a direct
association between huntingtin and the αC subunit of AP-2 has been reported
(39). HIP1R colocalizes more precisely with clathrin and with AP-2 in several cell
types (28) and the link between the HIP protein family and endocytosis is
validated by Sla2p, the S. cerevisiae orthologue of HIP1/HIP1R. Sla2p regulates
endocytosis and stabilizes actin organization (30,31,40). Like HIP1, HIP1R is also
enriched in brain clathrin-coated vesicles (28) but the region of HIP1 we identify
here to interact directly with the core endocytic machinery is notably absent from
HIP1R (Fig. 1A). The conserved sequence LFDQTF might facilitate binding to the
α appendage, but neither an adjacent LMDMD nor a DPF is present in HIP1R.
The sequence 348LIEIS in HIP1R does resemble the β-arrestin 2 type I clathrin-box
sequence 374LIEFE and could possibly confer on HIP1R the capacity to bind to the
clathrin heavy chain β-propeller. Alternatively, as the central coiled-coil domain
found in both proteins likely mediates dimerization (31,40) and, as there is some
evidence for an interaction between HIP1 and HIP1R (29), heterodimers of HIP1
and HIP1R, like amphiphysin I and II, may exist.
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The precise role of the AP-2 and clathrin binding sequences imbedded within
many endocytic accessory proteins is a matter of debate (2,32). Instead of simply
targeting accessory proteins to an existing clathrin bud site, our findings and
others (13) indicate rather that when linked to a lipid-binding module, tandemly
arrayed AP-2- and clathrin-binding sequences could play a more critical role in
clathrin-coat biogenesis. Because accessory proteins like AP180, epsin and HIP1
bind to clathrin directly in the absence of AP-2, these proteins could initiate
lattice assembly in a phosphoinositide-dependent fashion. S. cerevisiae epsin
orthologues Ent1p and Ent2p both have essential ENTH domains and clathrin-
binding motifs (41), so the coat promoting properties of the accessory proteins
could explain the surprising presence of clathrin-coated vesicles and the viability
of yeast strains engineered to lack all functional adaptor complexes and AP180
(42,43). Clustered recruitment of AP-2 together with or augmented by AP180,
epsin and/or HIP1 will provide a network of clathrin-binding sequences for local
amplification of clathrin lattice assembly. By linking physiological clathrin
recruitment and assembly to phospholipid metabolism, these accessory proteins
allow for precise control of the intracellular location of bud assembly and also
link the process to multiple regulatory inputs. Strikingly, while amphiphysin
also displays centrally located AP-2 and clathrin binding sequences (6,12,24)
analogous to epsin, AP180 and HIP1, an ENTH domain is not present in this
protein. Nevertheless, the N-terminal segment (the BAR domain; Bin1,
amphiphysin, Rvs161/167) of amphiphysin I displays inherent phospholipid
binding properties (44).
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The diversity of lipid-dependent coat-assembly regulators might possibly
facilitate the assembly of functionally different clathrin coats at the cell surface
(45) or impart discrete functions to a single assembling lattice. The carboxyl-
terminal I/LWEQ domain of HIP1 is 70% identical to HIP1R and 34% identical to
Sla2p, proteins known to bind to actin filaments (28,46), so it seems probable that
HIP1 also associates directly with assembled actin. In yeast, a temperature
sensitive mutant allele of Sla2p (end4-1) was identified in a screen for endocytic
mutants (30) but the I/LWEQ domain does not appear necessary to sustain
endocytosis (31). This is in line with our observation that the coat assembly
properties of mammalian HIP1 reside within the first 533 amino acids.
Nonetheless, positioned within the assembling lattice, the I/LWEQ domain in
HIP1 could link the clathrin-bud site to the adjacent actin cytoskeleton. Given the
enrichment of HIP1 in isolated clathrin coated vesicles, this interaction could
potentially explain the dramatic increase in the radial mobility of GFP-clathrin-
marked bud sites at the plasma membrane after depolymerization of the actin
cytoskeleton with jasplakinolide (47). The ability of HIP1 to bind to
phosphoinositides and actin simultaneously could also be linked to the
observation that in neurons derived from synaptojanin 1 knock-out animals,
clathrin-coated vesicles accumulate at the nerve terminal enmeshed within an
actin-like matrix (48). Irrespective, the modular arrangement of proteins like
AP180, epsin, amphiphysin and HIP1 appears to dictate related function in
clathrin-coat assembly.
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Acknowledgements
We are grateful to Stuart Kornfeld and Gerry Apodaca for constructive
comments on our manuscript and to Ora Weisz for the COS-7 cells and for help
with the transfection procedure. We also thank Juan Bonifacino, Pietro De
Camilli, Reinhard Jahn and Ernst Ungewickell for providing important reagents.
This work was supported in part by NIH grants RO1 DK53249 (L.M.T.), KO8
CA76025-01 (T.S.R.) and RO1 CA82363-01A1 (T.S.R.). T.S.R. is currently
supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell
foundation Award, DRS-22.
Footnotes
1. T.J. Brett, L.M. Traub and D.H. Fremont, manuscript submitted for publication.
2. If the density of the GST-αC appendage immobilized on GSH Sepharose is
reduced 5–10 fold, increasing the relative spacing of the individual appendages,
then the FXDXF-bearing SJ170C2 fragment is able to inhibit soluble eps15 and
epsin binding, T.J. Brett, D.H Fremont and L.M. Traub, unpublished
observations. This supports the notion that the cooperativity of the tandemly
arrayed DPF/W triplets in eps15 and epsin prevents efficient competition by the
HIP1M1 segment.
3. . While cytosolic clathrin binds avidly to the HIP1 GST fusions, we note that
HIP1M1 reproducibly interacts more efficiently with soluble trimers than the
HIP1M2 segment.
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Figure legends
Fig. 1. HIP1 structure and sequence alignment.
A. Domain organization of human (Hs) HIP1. The location of the FXDXF (red),
DPF (cyan) and clathrin-binding (green) sequences is indicated with vertical bars
while the regions corresponding to the GST-HIP1-fusion proteins used in this
study are shown below.
B. Local sequence alignment of human and mouse (Mm) HIP1 and HIP1R
sequences. Identical residues are colored pink and conservative
substitutions yellow. The locations of the FXDXF (red), DPF (cyan) and LMDMD
(green) motifs in HIP1 are indicated above.
Fig. 2. HIP1 is a component of clathrin-coated vesicles.
Aliquots of 20 µg each of rat brain homogenate, crude microsomes, cytosol and
purified clathrin-coated vesicles were fractionated by SDS-PAGE and either
stained with Coomassie blue (left panel) or transferred to nitrocellulose (right
panels) and probed with antibodies directed against the clathrin light chains
(LC), the µ2 subunit of the AP-2 complex, HIP1, AP180, epsin 1, amphiphysin, or
synaptotagmin I. The position of the molecular mass standards (in kDa) and the
~180-kDa clathrin heavy chain (HC) (left panel) are indicated on the left and
right respectively. Only the relevant portion of each blot (right panels) is shown.
Proteolysis of the clathrin light chains in the coated vesicle preparation suggests
the lower ~80-kDa band detected by the anti-HIP1 antibodies is likely a
degradation product.
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Fig. 3. HIP1 interacts with the core endocytic machinery.
A. Immobilized GST (lane a and b) or GST-SJ170C2 (lane c and d), GST-HIP1M1
(lane e and f), GST-HIP1M2 (lane g and h) or GST-HIP1M2 (DPF→KPS) (lane i
and j) was incubated with rat brain cytosol at 4°C for 60 min. After
centrifugation, aliquots of 1/80 of each supernatant (S) and 1/8 of each washed
pellet (P) were resolved by SDS-PAGE and either stained with Coomassie blue
(left panel) or immunoblotted (right panels) with antibodies directed against the
AP-2 α- or µ2 subunit or the clathrin heavy (HC) or light chain (LC).
B. Immobilized GST (lane a-d) or GST-αC appendage (lane e-h) were incubated
with either rat brain cytosol (lane a, b, e and f) or a rat brain detergent extract
(lane c, d, g and h). After centrifugation, aliquots of 1/80 of each supernatant (S)
and 1/8 of each washed pellet (P) were resolved by SDS-PAGE and either
stained with Coomassie blue (left panel) or immunoblotted (right panels) with
antibodies directed against HIP1, AP180 or epsin 1.
C. Immobilized GST (lane a and b) or GST-αC appendage (lane c-h) were
incubated with rat brain cytosol alone (lane a-d) or supplemented with 20 µM
epsin 1 DPW domain (lane e and f) or 20 µM HIP1M2 fragment (lane g and h).
After centrifugation, aliquots of 1/80 of each supernatant (S) and 1/8 of each
washed pellet (P) were resolved by SDS-PAGE and either stained with
Coomassie blue (left panel) or immunoblotted (right panels) with antibodies
directed against epsin 1, eps15, amphiphysin or AP180. The immunoreactive
signal below the epsin band after addition of the DPW domain (right panel, lane
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f) reflects strong reactivity of the antibody with this portion of epsin, to which it
was raised.
Fig. 4. A functional clathrin box within HIP1.
A. Immobilized GST (lane a and b) or GST-HIP1M1 (lane c and d) or GST-
HIP1M1 (LMD→AAA) (lane e and f) was incubated with rat brain cytosol. After
centrifugation, aliquots of 1/80 of each supernatant (S) and 1/8 of each washed
pellet (P) were resolved by SDS-PAGE and either stained with Coomassie blue
(left panel) or immunoblotted (right panels) with antibodies directed against the
AP-2 β-or µ2-subunit or the clathrin heavy (HC) or light chain (LC). Note that
using brain cytosol, the bound AP-2 complexes contain both the faster migrating
β2 subunit as well as lower amounts of the AP-1 β1 subunit (right panel, lane d
and f). The promiscuity of β subunit incorporation into AP-1 and AP-2 has been
described before (22).
Fig. 5. Effect of the overexpressed HIP1M1 segment on endocytosis.
COS-7 cells transiently expressing myc-tagged HIP1M1 for 48 hours were serum
starved for 60 min and then incubated with biotinylated transferrin for 15 min at
37°C prior to fixation. Endocytosed transferrin was visualized with streptavidin-
Alexa 594 (panel A, red) and the transfected cells identified with anti-myc mAb
9E10 and then an anti-mouse Alexa 488 conjugate (panel B, green). The merged
(panel C) image showing both endocytic inhibited (small arrows, no
accumulation of transferrin within perinuclear recycling endosomes) and
uninhibited (large arrowhead) cells is also shown.
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Fig. 6. Phosphoinositide-dependent clathrin-coat assembly.
A. Sequence alignment of the amino-terminal segments of rat (Rn) AP180 and
human HIP1 and HIP1R. Amino acid conservation is colored as in Fig. 1B. The
location of the first two α helices of the AP180 ENTH domain (13) is shown
above while the basic side chains required to coordinate PtdIns(4,5)P2 are
indicated below with vertical arrows.
B. Phosphoinositide-containing liposomes were first preincubated with HIP1
(1-533) (lanes c,d and g,h), AP-2 (lanes e-h and k,l) and GST-epsin (1-407) (lanes i-
l) at 4°C for 60 min as indicated. After recovery by centrifugation, each liposome
pellet was resuspended and then incubated at 4°C for 60 min with purified
clathrin trimers. After centrifugation, aliquots of 1/25 of each supernatant (S)
and 1/4 of each pellet (P) were resolved by SDS-PAGE and stained with
Coomassie blue.
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HIP1(1-533)HIP1M1HIP1M2
ENTH coiled coil I/LWEQ
Hs HIP1
1 1034
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Hs HIP1
305
ISPVVVIPAEA--------------SSPDSEPVLEKDDLMDMDASQQN
Mm HIP1
308
ISPVVVIPAEV--------------SSPDSEPVLEKDDLMDMDASQQT
Hs HIP1R
299
IKPVVVIPEEAPEDEEPENLIEISTGPPAGEPVVVAD-----------
Mm HIP1R
299
IKPVVVIPEEAPEEEEPENLIEISSAPPAGEPVVVAD-----------
Hs HIP1
339
LFDNKFDDIFGSSFSSDPFNFNSQNGVNKDEKDHLIERLYRE
Mm HIP1
342
LFDNKFDDVFGSSLSSDPFNFNNQNGVNKDEKDHLIERLYRE
Hs HIP1R
336
LFDQTF---------------GPPNGSVKDDRDLQIESLKRE
Mm HIP1R
336
LFDQTF---------------GPPNGSVKDDRDLQIENLKRE
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AB
C
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α1
α2
Rn AP180
17
VTGSAVSKTVCKATTHEVMGPKKKHLDYLIQATNE
Hs HIP1
32
SFERTQTVSINKAINTQEVAVKEKHARTCILGTHH
Hs HIP1R
26
QFDKTQAISISKAINTQEAPVKEKHARRIILGTHH
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and Linton M. TraubSanjay K. Mishra, Nicole R. Agostinelli, Tom J. Brett, Ikuko Mizukami, Theodora S. Ross
endocytic accessory proteinsClathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for
published online September 27, 2001J. Biol. Chem.
10.1074/jbc.M108177200Access the most updated version of this article at doi:
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