Human Molecular Genetics, 2001, Vol. 10, No. 17 1807-1817
© 2001 Oxford University Press
The huntingtin interacting protein HIP1 is a clathrin and 
-adaptin-binding protein involved in receptor-mediated endocytosis
1Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin (Dahlem), Germany, 1GPC Biotech AG, Fraunhoferstrasse 20, D-82152 Martinsried, Germany and 2Division of Medical and Molecular Genetics, United Medical and Dental Schools, Guy’s Hospital, London SE1 7EH, UK
Received April 17, 2001; Revised and Accepted June 21, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The huntingtin interacting protein (HIP1) is enriched in membrane-containing cell fractions and has been implicated in vesicle trafficking. It is a multidomain protein containing an N-terminal ENTH domain, a central coiled-coil forming region and a C-terminal actin-binding domain. In the present study we have identified three HIP1 associated proteins, clathrin heavy chain and
-adaptin A and C. In vitro binding studies revealed that the central coiled-coil domain is required for the interaction of HIP1 with clathrin, whereas DPF-like motifs located upstream to this domain are important for the binding of HIP1 to the C-terminal ‘appendage’ domain of -adaptin A and C. Expression of full length HIP1 in mammalian cells resulted in a punctate cytoplasmic immunostaining characteristic of clathrin-coated vesicles. In contrast, when a truncated HIP1 protein containing both the DPF-like motifs and the coiled-coil domain was overexpressed, large perinuclear vesicle-like structures containing HIP1, huntingtin, clathrin and endocytosed transferrin were observed, indicating that HIP1 is an endocytic protein, the structural integrity of which is crucial for maintenance of normal vesicle size in vivo. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by an elongated polyglutamine (polyQ) repeat (more than 37 glutamines) located at the N-terminus of a large
350 kDa protein, huntingtin (1). The pathomechanism of HD as well as the normal function of huntingtin are unclear. Both normal and mutated forms of huntingtin have been shown to be expressed in the central nervous system and peripheral tissues (2). Early studies revealed that huntingtin is predominantly a cytoplasmic protein, a fraction of which is associated with vesicles and microtubules, suggesting that it plays a functional role in the cell filament networks or the transport of vesicles (3–5). Velier et al. (6) showed by immunofluorescence microscopy and subcellular fractionations that huntingtin co-localizes with membranes of the trans-Golgi-network and also with clathrin-coated vesicles in the cytoplasm. To gain insight into the normal function of huntingtin several investigators have screened for huntingtin interacting proteins using the yeast two-hybrid system (for review see ref. 7). The first huntingtin associated protein discovered in this way was HAP1 (8). Like huntingtin, HAP1 has no homology to previously known proteins in the databases. However, similar to huntingtin, HAP1 is expressed predominantly in neurons and associated with microtubules, membranous organelles and synaptic vesicles (9–11). After the discovery of HAP1, the huntingtin interacting protein (HIP1) has been identified by two-hybrid screening (12,13). HIP1 is predominantly expressed in brain, but can also be detected in peripheral tissues at low levels. Subcellular fractionations revealed that both HIP1 and huntingtin are enriched in membrane-containing fractions, suggesting that this protein, similar to huntingtin, plays a functional role in vesicle transport in vivo (12). HIP1 is the human homolog of Saccharomyces cerevisiae Sla2p, also known as End4p and Mop2p (14–16). Immunolocalization studies in yeast have shown that Sla2p is a component of the cortical actin cytoskeleton (17). Furthermore, functional studies revealed that this protein is required for the internalization step of endocytosis (14). For endocytosis in yeast, the central coiled-coil domain but not the C-terminal talin-like domain of Sla2p is essential. Other homologs of HIP1 have been identified in nematodes, Drosophila, mouse and humans (15,18,19), but little is known about their cellular function. Recently, Engqvist-Goldstein et al. (19) have shown that the mouse protein mHip1R co-localizes with clathrin, AP-2 and endocytosed transferrin in mammalian cells, implying that this protein, similar to huntingtin, plays a role in clathrin-mediated endocytosis. However, a direct interaction between HIP1 and components of clathrin-coated vesicles has not been demonstrated.
To learn more about the cellular function of HIP1 and huntingtin, we have searched for novel HIP1-interacting proteins using affinity chromatography. In this study, we show that HIP1 directly associates with clathrin heavy chain as well as -adaptin A and C. These proteins are major components of clathrin-coated vesicles and are important for receptor-mediated endocytosis at the plasma membrane. We also found that HIP1 contains an Asp-Pro-Phe (DPF) motif, and that this motif is important for the interaction of the protein with the ‘appendage’ domains of -adaptin A and C. Furthermore, in vitro binding experiments revealed that a predicted coiled-coil forming domain located downstream of the DPF motif is critical for the association of HIP1 with clathrin heavy chain. Together, these findings strongly support the hypothesis that HIP1 and huntingtin play a functional role during endocytosis and synaptic vesicle recycling in neurons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Affinity purification of HIP1-binding proteins
To find new clues about the function of HIP1 we first examined the domain structure of the protein using the SMART and Pfam programs (Fig. 1). At the N-terminus of HIP1, a highly conserved epsin N-terminal homology (ENTH) domain of approximately 120 amino acids was identified. This domain has been detected previously in several proteins involved in endocytosis and the regulation of the actin cytoskeleton, including AP180 (20), CALM (21) and epsin (22). Recently, it has been demonstrated that the ENTH domain of CALM binds the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), and based on the close similarity of the ENTH domains of CALM and HIP1, it has been proposed that HIP1 also associates with PIP2-containing membranes via its N-terminus (23,24). Downstream of the HIP1 ENTH domain, there are four copies of the sequence motif DxF (where x is R, L, I or P) followed by a central coiled-coil forming domain containing the potential clathrin-binding motif, VDLE. DPF/W motifs have been identified in a number of proteins that play a functional role in endocytosis (e.g. AP180, amphiphysin, Eps15 and epsin), and in all of these proteins the DPF/W motif has been implicated in the binding to the appendage domain of
-adaptin, a component of the adaptor protein (AP) complex present in clathrin-coated vesicles (25,26). A talin-like actin-binding domain of approximately 200 amino acids is located at the C-terminus of HIP1 and appears to link the protein to F-actin filaments (19).
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In order to determine whether HIP1 specifically associates with proteins involved in endocytosis, a truncated HIP1 fragment (residues 218–604, Fig. 1) containing the DPF-like motifs and the predicted coiled-coil forming domain was expressed as glutathione S-transferase (GST) fusion in Escherichia coli and affinity purified on glutathione agarose beads (27). Protein extract prepared from human cortex was then incubated with the GST-HIP1 (218–604) affinity matrix in the presence of protease inhibitors. After extensive washing of the beads, bound proteins were eluted with SDS-sample buffer and analyzed by SDS–PAGE and Coomassie blue staining. As shown in Figure 2A, three prominent proteins migrating on an 8% SDS gel at
170, 110 and 105 kDa were retained by the GST-HIP1 affinity matrix (lanes 1–3), but not by the control beads containing bound GST (lane 5) or the beads without any immobilized protein (lane 4). The input GST-HIP1 (218–604) fusion protein migrating in the SDS-gel at 75 kDa was detected by immunoblotting using anti-GST antibody (data not shown). The enrichment of the three HIP1-associated proteins from human brain extract by affinity chromatography was reproducibly observed in at least three pull-down experiments using different extract preparations. The same result was obtained using the GST-HIP1 (1–604) fragment as a bait (data not shown).
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Identification of HIP1 interacting proteins by mass spectrometry and immunoblotting
For identification of the isolated GST-HIP1 (218–604)-interacting proteins the protein bands at 170, 110 and 105 kDa were excised from Coomassie blue-stained gels, ‘in-gel’ digested with trypsin, and the recovered cleavage products were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). As an example, the MALDI-MS peptide map of the 170 kDa band is shown in Figure 2B. A search of the peptide masses in the SwissProt sequence database showed that 35 matched the calculated tryptic peptide masses of human clathrin heavy chain 1 (GenBank accession no. Q00610) covering 30% of the sequence. The complete results of the MALDI-MS analysis are summarized in Table 1. The proteins corresponding to the 110 and 105 kDa bands were identified unambiguously as
-adaptin A and -adaptin C, respectively, which like clathrin are key components of the clathrin-coated pits and vesicles. Clathrin heavy chains provide the structural framework for the polyhedral lattice that is believed to be the driving force behind membrane invagination and the formation of clathrin-coated vesicles (28). The -adaptin proteins A and C are components of the AP-2 adapter complex that triggers the assembly of the clathrin lattice at the plasma membrane. AP-2 complexes interact with cytoplasmatically exposed sorting signals and presumably play a direct role in concentrating cargo molecules in coated pits (29).
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The identity of clathrin heavy chain and
-adaptin A and C was confirmed by immunoblotting using antibodies specifically directed against clathrin heavy chain and -adaptin (Fig. 1C). Furthermore, western blot analysis revealed that huntingtin or some N-terminal fragments thereof, as well as the huntingtin interacting protein HAP1 were retained by the GST-HIP1 (218–604) affinity matrix (lanes 1–3). Again, none of these proteins was retained by the control GST beads (lane 5) or the empty glutathione agarose beads (lane 4). In a separate experiment we found that dynamin, a GTPase which mediates the liberation of nascent clathrin-coated vesicles from the plasma membrane (30), was also selectively enriched by the GST-HIP1 beads (data not shown). Together, these results suggest that huntingtin and its interacting proteins HIP1 and HAP1, and possibly also dynamin, are all part of a large protein complex that is associated with clathrin-coated vesicles.
HIP1 fragments containing the DPF-like motifs directly associate with the -adaptin appendage domain
Recent studies have identified several proteins that bind to the C-terminal appendage domain of -adaptin, including the amphiphysins, AP180, auxilin, Eps15 and epsin (26). The only common feature among these protein sequences is that they contain either the DPF or DPW motifs. In order to test whether HIP1, containing a single DPF and three DxF motifs, is capable of interacting with the -adaptin appendage domain, pull-down experiments from brain cytosol were performed using a GST-tagged -adaptin C appendage domain (C) as a bait. As shown in Figure 3A, full length HIP1 was selectively retained by the GST-C affinity matrix along with amphipysin, which like HIP1 contains a single DPF motif at the N-terminus. Identical results were obtained using the appendage domain of -adaptin A as a bait (data not shown).
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In order to establish a direct interaction between HIP1 and the
-adaptin appendage domain, GST-C was used as a probe in overlay assays of recombinant His-HIP1 (218–604) expressed in E.coli. Figure 3B shows that GST-C selectively binds to the immobilized His-HIP1 (218–604) protein, whereas GST alone does not. No such interaction was observed with His-HIP1 (1–217) in which the region containing the DxF motifs is lacking (data not shown). Thus, HIP1 can now be considered as a binding partner of both -adaptin A and C.
To further investigate the nature of the interaction between HIP1 and -adaptin, in vitro binding experiments with recombinantly expressed His-HIP1 (1–604) and GST-C in the presence or absence of competing peptides were performed. Peptides corresponding to the HIP1 DPF region (SSFSSDPFNFNS) or to the SH3GL3 SH3 domain (CQLPQPPPQAQPLLPQPQ) (31) were used for the competition assays. As shown in Figure 3C, only the DPF peptide, but not the SH3 control peptide, disrupted the HIP1--adaptin interaction, indicating that as for other proteins involved in endocytosis (e.g. Eps15, epsin, AP180) the DPF motif in HIP1 plays a role in mediating the binding of the protein to the -adaptin appendage domain.
The coiled-coil forming domain in HIP1 is critical for the binding to clathrin heavy chain
To map the region in HIP1 which is responsible for its binding to clathrin heavy chain, pull-down experiments from human brain extract using various GST-HIP1 fusions were performed. As shown in the immunoblot of Figure 3D, clathrin heavy chain was pulled down by the GST-HIP1 fusions 1–604, 218–604 and 334–604, but not by the GST-HIP1 fusions 1–333 and 1–217, indicating that the potential coiled-coil forming domain (residues 334–604), but not the region containing the DxF motifs (residues 218–333) is important for the interaction of HIP1 with clathrin. When the same blot was probed with anti--adaptin A antibody, a different result was obtained. Only the GST-HIP1 fusions 1–604, 1–333 and 218–604 containing the DxF motifs bound -adaptin A, whereas the GST-HIP1 fusions 1–217 and 334–604, lacking this sequence motifs, did not. Thus, while HIP1 associates with the endocytic adaptor protein complex via its DPF-like motifs, the coiled-coil forming domain located immediately downstream of these motifs appears to be crucial for its binding to clathrin heavy chain.
HIP1 is associated with clathrin-coated vesicles
Previously, we have shown by subcellular fractionation of human cortex that HIP1 and huntingtin immunoreactivities are enriched in the membrane-containing fractions (12). To examine whether full length HIP1 is directly associated with clathrin-coated vesicles, vesicle-containing membrane fractions were purified from human cortex and analyzed by SDS–PAGE, immunoblotting and electron microscopy. Coomassie blue staining and western blot analysis revealed that clathrin heavy chain, migrating in SDS-gels at about 170 kDa, is highly enriched in the purified vesicle fraction (Fig. 4A and B). Besides clathrin, huntingtin, HIP1, HAP1 and -adaptin A and C were found to be enriched in this fraction (Fig. 4B), whereas BiP, a protein known to be resident in the ER (32), was barely detectable. Analysis of the purified membrane fraction by transmission electron microscopy showed numerous clathrin-coated vesicles with the characteristic triskelion shape (Fig. 4Ca). Immunolabeling of the vesicles with specific antibodies revealed that in addition to clathrin heavy chain (Fig. 4Cc) and -adaptin (Fig. 4Cd) the proteins HIP1, HAP1 and huntingtin (Fig. 4Cb–f) were directly associated with the vesicles, in agreement with the western blotting results.
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Overexpression of HIP1 (218–604) in COS-1 cells induces the formation of large vesicle-like structures
To examine whether HIP1 and clathrin co-localize in vivo, human fibroblasts were analyzed by immunofluorescence microscopy. Cells were permeabilized and double-labeled with anti-HIP1 and anti-clathrin antibodies. HIP1 and clathrin appeared to overlap mainly in the perinuclear region and in punctate vesicle-like structures dispersed throughout the cytoplasm. However, only a portion of the clathrin-positive puncta co-localized with HIP1 staining, indicating that not all clathrin-coated vesicles may contain the HIP1 protein (Fig. 5A–C). Similar results were obtained when non-transfected COS-1 cells were double-labeled with anti-HIP1 and anti-clathrin heavy chain antibodies or when the full length human HIP1 protein (residues 1–1003) was overexpressed in these cells (data not shown). These results are consistent with previous observations in COS-7 cells overexpressing the full length mouse Hip1R protein (19). Surprisingly, when the truncated HIP1 (218–604) fragment, containing the DPF-like motifs and the adjacent coiled-coil domain, was overexpressed in COS-1 cells, formation of large perinuclear vesicle-like structures was observed. These structures contained in addition to HIP1 protein, clathrin heavy chain and huntingtin endocytosed transferrin (Fig. 5D–L), suggesting that they are produced during the process of clathrin-mediated endocytosis. Similar results were obtained when the truncated HIP1 fragment was overexpressed in 293 and HeLa cells (data not shown). Overexpression of the truncated HIP1 protein which lacks the ENTH and talin-like domains apparently interferes with this process, resulting in the accumulation of oversized vesicle-like structures in the perinuclear region. We conclude that HIP1 is an endocytic protein and that its structural integrity is crucial for the maintenance of a uniform-size vesicle population in the cell.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntingtin and its interacting partner HIP1 have been implicated in endocytosis and vesicle trafficking (12), yet until today no direct links between these proteins and components of the endocytic machinery have been established. Here we show for the first time that HIP1, a homolog of yeast Sla2p, directly associates with clathrin-coated vesicles. Sequence alignments and domain structure predictions revealed that HIP1 is a modular protein with several conserved domains that could potentially interact with other endocytic proteins. At the N-terminus of HIP1 there is a highly conserved region termed the ENTH domain. ENTH domains are structural modules of approximately 140 amino acids typically found in proteins involved in clathrin-mediated endocytosis such as epsin, AP180 and CALM and their respective yeast homologs (33). Recently, two independent groups have demonstrated a strong affinity of the close AP180 homolog, CALM and epsin for PIP2, implying that these proteins directly bind to lipid membranes via their conserved ENTH domains (24,23). Both AP180 and epsin are known to play important roles in clathrin-mediated endocytosis. In addition to PIP2, AP180 also binds to clathrin and stimulates clathrin cage assembly in an in vitro budding assay, limiting the size distribution of the resulting cages (34). Thus, the simultaneous binding of AP180 to PIP2 and clathrin may serve to tether clathrin to membrane vesicles and help to determine the size of the resulting clathrin-coated vesicles. At present, the function of the ENTH domain in HIP1 is unknown but, based on its close sequence similarity to the ENTH domains of AP180 and CALM, it is very likely that HIP1 also binds to membrane vesicles via its N-terminal domain. Our data showing that HIP1 is associated with clathrin-coated vesicles is consistent with this view (Fig. 4).
Pull-down experiments from human brain cytosol have shown that HIP1 can bind to -adaptin, a subunit of the clathrin adapter complex AP-2, and the region of HIP1 that binds -adaptin has been mapped to residues 218–333 (Fig. 3D). This region is characterized by the presence of three copies of the tripeptide DxF (where x is R, L or I) and a single DPF triplet typically found in multiple copies in endocytic proteins such as Eps15, epsin, AP180 and auxilin that interact with the ‘appendage’ of -adaptin. That HIP1 directly binds to the -adaptin appendage domain was confirmed by in vitro binding experiments using a recombinantly expressed C appendage domain protein. Furthermore, a DPF-containing peptide of HIP1 (residues 319–330) was found to inhibit its binding to the C appendage domain, supporting the notion that the DPF motifs (and perhaps also the DxF motifs) present in appendage domain binding partners play a crucial role in their interaction with the domain (26,25). It should be noted that the same region in HIP1 that contains the four DxF sequence motifs also contains four NxF repeats (where x is N, L or K). At present the function(s) of these tripeptides is unclear, but it is well known that NPF motifs are ligands for the EH domains of Eps15 (22).
Interestingly, the same region in HIP1 that contains the DxF and NxF sequence motifs is also required for the binding of the protein to huntingtin (12). The N-terminus of huntingtin contains a HEAT repeat which is most likely involved in the protein–protein interaction with HIP1. HEAT repeats are protein–protein interaction modules composed exclusively of -helices that were found in a variety of proteins involved in cellular transport processes (35). Although the sequence motifs in HIP1 important for huntingtin binding are not known, our data clearly show that huntingtin can be enriched from human brain cytosol using a GST-HIP1 (218–604) affinity matrix. Furthermore, huntingtin was found to be associated with isolated clathrin-coated vesicles, indicating that a stable protein complex consisting of huntingtin, HIP1, AP-2 and clathrin may exist in neuronal cells. Using yeast two-hybrid experiments, it has been shown previously that the huntingtin–HIP1 interaction is inversely correlated to the length of polyglutamine tract in huntingtin (13). Thus, disruption of the normal huntingtin–HIP1 interaction by elongated polyglutamine sequences may contribute to the disease phentotype in HD patients. However, additional studies are necessary to determine whether elongated polyglutamine sequences in huntingtin will impair the formation and/or the transport of clathrin-coated vesicles in vivo.
In HIP1, the region with the DxF motifs is immediately followed by a putative coiled-coil forming domain (Fig. 1). Coiled-coils are -helical motifs that mediate subunit oligomerization of a large number of cytoskeletal and motor proteins (36). We found that the coiled-coil domain in HIP1 is required for its binding to clathrin heavy chain. Sequence comparisons revealed that this segment of HIP1 contains a VDLE sequence motif that closely resembles the consensus residues, L(L,I,V)(D,E,G,N)(L,F)(D,E,Q), for clathrin binding (33) and thus could potentially serve as the ligand for clathrin heavy chain.
Our pull-down experiments with GST-HIP1 (218–604) revealed that besides clathrin and -adaptin A and C, the huntingtin interacting protein HAP1 (8) was also selectively enriched from brain cytosol. HAP1 and huntingtin have been previously shown to be associated with microtubules and synaptic vesicles, suggesting that these proteins link vesicles to microtubules. Also, there is evidence to indicate that HAP1 binds to p150Glued, a subunit of the dynactin complex (37,38). Dynactin is associated with cytoplasmic dynein, an ATP-dependent microtubule motor protein involved in vesicle retrograde transport (39). Thus, a protein complex consisting of HAP1, huntingtin and p150Glued may link clathrin-coated vesicles to dynein motor proteins. In support of this hypothesis, fast retrograde movement of huntingtin and HAP1 in axons has been reported (40). Like HIP1, HAP1 contains a central coiled-coil forming domain (41), and preliminary experiments in our laboratory using the yeast two-hybrid system revealed that HIP1 and HAP1 interact with each other via their central coiled-coil domains (H.Goehler and E.E.Wanker, unpublished data). A leucine zipper present within the central region of both HIP1 and HAP1 is likely to facilitate their interaction.
Recently, Engqvist-Goldstein et al. (19) have shown that mHipR, the mouse homolog of hHIP1R, binds to F-actin via the C-terminal talin-like domain. Furthermore, they demonstrated co-localization of full length mHip1R with punctate uniform-size clathrin-coated vesicles distributed throughout the cytoplasm. Our results obtained with full length HIP1 in COS-1 cells are consistent with these observations and indicate that human HIP1 is also associated with normal-size clathrin-coated vesicles in vivo. However, when a truncated HIP1 fragment (residues 218–604) lacking both the N-terminal ENTH and the C-terminal talin-like domains was overexpressed, formation of large perinuclear vesicle-like structures in the perinuclear region was observed. These structures contained HIP1, huntingtin and clathrin. Furthermore, uptake assays revealed that these large vesicle-like structures also contained Texas red-labeled transferrin, suggesting that they are formed in the course of clathrin-mediated endocytosis. Interestingly, no such oversized vesicles were formed when HIP1 fragments lacking either the N-terminal ENTH (residues 218–1003) or the C-terminal talin domain (residues 1–604) were overexpressed in COS-1 cells (S.Waelter, unpublished data), indicating that the presence of either of these domains in HIP1 is sufficient for preventing the production of the abnormal perinuclear vesicle-like structures.
A model of the potential role of HIP1 in endocytosis is shown in Figure 6. As viewed by microscopy, endocytosis begins with the formation of a lattice, composed of clathrin triskelions, at the cytosolic face of the plasma membrane. This lattice recruits receptors and other cargo molecules into clathrin-coated pits which then bud off as vesicles. The clathrin adaptor complex AP-2, a heterotetramer composed of , ß, µ and 
subunits, and AP180, an ENTH domain containing protein, are known to stimulate the assembly of the clathrin coat and subsequent vesicle budding (42,43). AP180 and AP-2 bind with high affinity to the membrane lipids PIP2 and phosphatidylinositol-3,4,5-trisphosphate (PIP3), respectively, and the interaction of these proteins with phoshoinositides appears to be important for anchoring the clathrin coat to the plasma membrane (23,44,45). We propose that HIP1 similar to AP180 and AP-2 is also involved in the recruitment of clathrin coats to lipid membranes. Once bound to the complex consisting of AP-2, AP180 and HIP1, clathrin begins to polymerize and drives the invagination of the coated pits. Assembly of dynamin into spiral collars at the neck forms constricted coated pits and triggers vesicle budding (46). During this process HIP1 links the clathrin-coated vesicles to the actin cytoskeleton and thereby controls the size of the nascent endocytic vesicles. Thus, in neuronal cells, HIP1 may function as an AP that is required for the spatial organization of clathrin-coated vesicles. In addition, it may participate in the transport of endocytic vesicles to target organelles in the cell. A detailed understanding of how HIP1 participates in the various steps of clathrin-coat assembly, vesicle budding, detachment of the newly formed vesicles and their movement away from the plasma membrane into the cytosol not only may help to elucidate key steps in endocytosis and vesicle trafficking, but may also provide new clues to the normal function of huntingtin and its interacting partner HAP1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies
Commercially available antibodies were anti-clathrin heavy chain (HC) polyclonal antibody C-20 (Santa Cruz), anti-clathrin monoclonal antibody (Biodesign), anti-clathrin monoclonal antibody (Transduction Laboratories), anti-GST polyclonal antibody (Amersham Pharmacia), anti-
-adaptin polyclonal antibody A-19 (Santa Cruz), anti--adaptin A monoclonal antibody (Transduction Laboratories), anti-amphiphysin monoclonal antibody (Transduction Laboratories), anti-BiP polyclonal antibody (Transduction Laboratories) and monoclonal RGS-His antibody (Qiagen). The polyclonal huntingtin-specific antibody CAG53b (47), the affinity-purified polyclonal anti-HIP1 antibody H24 (12) and the polyclonal huntingtin-specific antibody HD1 (47) have been described. Polyclonal anti-HAP1 antibody was a kind gift of C.Ross, and the monoclonal anti-huntingtin antibody 4C8 was a kind gift of J.-L.Mandel.
Plasmid construction
To produce GST-tagged HIP1 (residues 218–604), an 1.2 kb HIP1 cDNA fragment was released from pQE32-HIP1 (12) and subcloned into pGEX-5X-1 (Pharmacia). A construct encoding the full length HIP1-2 protein (residues 1–1003, hereafter referred to as HIP1) was kindly provided by M.Hayden. The cDNAs encoding residues 1–604, 1–333, 1–217 and 334–604 were amplified from the HIP1 full length plasmid by PCR and subcloned into the vector pGEX-6P-1 (Pharmacia) for production as N-terminal GST fusion proteins, into pQE32 (Qiagen) for production as N-terminal His6-tagged fusion proteins, and into pTL1-HA2 (31) for eukaryotic expression as N-terminal hemagglutinin-tagged fusion proteins under the control of a SV40 promotor. DNA encoding residues 701–938 of mouse -adaptin C (the appendage domain) was obtained by PCR using the full length -adaptin C cDNA (48) and subcloned into pGEX-6P-1 for production as an N-terminal GST fusion protein.
Protein expression and purification
GST fusion proteins and N-terminal His6-tagged fusions were expressed in E.coli SCS1 (pSE111) (49) at 25°C overnight and affinity purified on glutathione-agarose (Sigma) or Ni-NTA-agarose (Qiagen), respectively, using standard protocols (12,27). For use in binding experiments, the proteins were dialyzed against 25 mM HEPES pH 7.5, 150 mM NaCl, 4 mM dithiothreitol (buffer A) and stored at 0–4°C.
Protein–protein binding assays
Human brain extract was prepared by homogenizing 1 g human cortex in 1.5 ml lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 20 mM NaF, 10% glycerol, 1% NP40) with protease inhibitors and removal of the insoluble material by centrifugation at 16 000 g for 30 min. Unless otherwise noted, binding assays were performed by incubating 25 µg GST or GST fusion proteins, immobilized on 15 µl packed glutathione agarose in 5.3 mg/ml human brain cytosol or 1 mg/ml bacterial extract containing expressed His6-tagged fusion proteins for 60 min at 4°C. All assays were performed in buffer A containing 0.1% Triton X-100 in a final volume of 0.5 ml. The agarose beads were recovered by centrifugation at 10 000 g for 1 min, washed three times with 0.5 ml ice-cold buffer A, and the bound material was analyzed by SDS–PAGE and western blotting.
Protein overlay assay
Whole-cell extract of E.coli containing recombinantly expressed His6-HIP1 (218–604) (1 µg/lane) was subjected to SDS–PAGE, and proteins were transferred onto nitrocellulose. After blocking in 5% milk, the blots were incubated overnight at 4°C with 2.5 µg/ml GST or GST--adaptin C appendage domain (C) in 5% calf serum. After washing extensively, bound GST-C was detected with an antibody raised against GST.
Purification of clathrin-coated vesicles
Clathrin-coated vesicles were purified from human cortex as described by Lindner et al. (50). Briefly, 15 g of human cortex were homogenized in a Potter–Elvehjem device in 50 ml of buffer B (0.1 M MES, 0.5 mM MgCl2, 1mM EGTA, 0.02% NaN3, pH 6.5), supplemented with 0.1 mM PMSF prior to use, and centrifuged at 8000 g for 50 min. The resulting postmitochondrial supernatants were collected and spun at 100 000 g for 1 h, resulting in a microsomal pellet and the supernatant S1. The microsomal pellets were resuspended with 1–2 vol of buffer B. After extensive homogenization, the microsomes (P2) were mixed with the same volume of 12.5% ficoll-sucrose solution and centrifuged at 43 000 g for 40 min, generating pellet P3. The supernatant containing clathrin-coated vesicles was diluted with 4 vol. of buffer B containing 0.1mM PMSF and subsequently spun at 100 000 g for 1 h. The resulting pellet was resuspended in 100 µl buffer B containing 0.1 mM PMSF and homogenized (V). Samples were analyzed by SDS–PAGE followed by Coomassie blue staining and western blotting.
Mass spectrometry
Tryptic peptide mass maps of the proteins were recorded on a Bruker Scout MTP Reflex III MALDI mass spectrometer (Bruker Daltonik, Germany) using the matrix -cyano-4-hydroxycinamic acid.
Immunofluorescence and immunogold labeling
Fibroblasts were grown in flasks and prepared for immunofluorescence as described by Sittler et al. (31). COS-1 cells were transfected with pTL1-HIP1 constructs using calcium phosphate. Cells were fixed in freshly prepared paraformaldehyde and stained with appropriate primary and secondary antibodies following standard protocols. For the study of transferrin uptake, COS-1 cells expressing HIP1 (218–604) were starved at 37°C in serum-free medium and then incubated for 1 h in serum-free medium containing 25 mg/ml Texas red-labeled transferrin (Molecular Probes). For immunogold labeling the purified clathrin-coated vesicles were incubated for 2 h with primary antibodies. After centrifugation at 100 000 g for 10 min, the resuspended vesicles were incubated for 4 h with secondary (5 or 10 nm) immunogold antibody (British BioCell) and negatively stained with 2.5% ammonium molybdate for electron microscopic analysis.
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ACKNOWLEDGEMENTS |
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We thank M.Hayden for providing full length HIP1 cDNA, M.Robinson for providing full length
-adaptin A and C cDNAs, C.Ross for providing anti-HAP1 antibody, J.-L.Mandel for providing 4C8 antibody, M.Kelly for providing the SH3-domain containing peptide and S.Schnögl for reading the manuscript. This work was supported by the Max Planck Gesellschaft and grants from the Huntington’s Disease Society of America, HFSP, Deutsche Forschungsgemeinschaft (W1151/1-1) and the BMBF (BioFuture project: 0311853).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: + 49 30 8413 1351; Fax: +49 30 8413 1380; Email: wanker@molgen.mpg.de
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Y. Li, L.-S. Chin, A. I. Levey, and L. Li Huntingtin-associated Protein 1 Interacts with Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate and Functions in Endosomal Trafficking J. Biol. Chem., July 26, 2002; 277(31): 28212 - 28221. [Abstract] [Full Text] [PDF]
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V. Legendre-Guillemin, M. Metzler, M. Charbonneau, L. Gan, V. Chopra, J. Philie, M. R. Hayden, and P. S. McPherson HIP1 and HIP12 Display Differential Binding to F-actin, AP2, and Clathrin. IDENTIFICATION OF A NOVEL INTERACTION WITH CLATHRIN LIGHT CHAIN J. Biol. Chem., May 24, 2002; 277(22): 19897 - 19904. [Abstract] [Full Text] [PDF]
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