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Human Molecular Genetics Pages 2205-2212

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin
Introduction
Results
Discussion
Materials And Methods
   Yeast two-hybrid constructs and screening
   Cell culture and transfection of HEK 293 cells
   Generation of HAP1 antibody (AP157)
   Western blot analysis
   In vitro binding assay
   Immunoprecipitation
   Subcellular fractionation
   Immunocytochemistry
   GenBank accession number
Acknowledgements
Disclosure
Abbreviations
References
Note Added In Proof

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin

Huntingtin-associated protein 1 (HAP1) interacts with the p150 Glued subunit of dynactin Simone Engelender1, Alan H. Sharp1, Veronica Colomer1, Mariko K. Tokito5, Anthony Lanahan2,3, Paul Worley2,3, Erika L.F. Holzbaur5 and Christopher A. Ross1,2,4,*

Departments of 1Psychiatry, 2Neuroscience and 3Neurology and 4Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA and 5Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104-6046, USA

Received May 30, 1997; Revised and Accepted September 13, 1997

DDBJ/EMBL/GenBank accession no. AF010146

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150Glued. Binding of HAP1 to p150Glued (amino acids 879-1150) was confirmed in vitro by binding of p150Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150Glued from brain extracts, indicating that the interaction occurs in vivo. Like HAP1, p150Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.

INTRODUCTION

Huntington's disease (HD) is a progressive neurodegenerative illness inherited in an autosomal dominant manner (1 ,2 ). The gene which causes HD was cloned using a positional cloning approach and the mutation is an expanding CAG repeat coding for polyglutamine (3 ). The HD gene codes for a large protein of unknown function and without significant homology to any known protein in the database. Normal individuals have between five and ~30 consecutive glutamines in huntingtin while patients with the disease have >= 36 consecutive glutamines. The longer the expansion within the abnormal range, the earlier the age of onset of the illness (4 -6 ). The protein is widely expressed in both brain and peripheral tissues, but is enriched in neurons (7 -10 ).

Huntingtin has a widespread distribution within neurons, including cell bodies, dendrites, axons and nerve terminals. It has both a particulate and soluble localization and is enriched in the crude synaptic vesicle fraction (7 ,8 ). Detailed subcellular fractionation studies and electron microscopic studies have suggested a localization to cytoskeletal elements and vesicles within cells (7 -10 ).

We and several other groups have attempted to identify proteins which interact with huntingtin, as a clue to its normal function and its possible role in the disease (11 -15 ). Using the yeast two-hybrid system we previously identified a protein, huntingtin-associated protein (HAP1), which binds to huntingtin in a glutamine repeat length-dependent manner, suggesting that the interaction between huntingtin and HAP1 could be implicated in the pathogenesis of the disease (11 ). However, HAP1, like huntingtin, has no homology to previously known genes in the database. The HAP1 protein is also expressed in neurons and is enriched in a nerve terminal vesicle-rich fraction (16 ). In order to better understand the molecular interactions of HAP1, we have now sought to determine its protein interaction partners. Recently we have shown that the N-terminus of HAP1 interacts with a protein we have termed Duo (17 ). We now report that HAP1 interacts with the p150Glued subunit of dynactin and with the pericentriolar autoantigen protein PCM-1.

RESULTS

In order to use the HAP1 cDNA as a probe in the yeast two-hybrid system, the cDNA was divided into three overlapping subclones (Fig. 1 A). We have previously found two cDNAs which appeared to arise by alternative splicing, termed HAP1-A and HAP1-B (11 ). Therefore, two constructs were made from the C-terminus. The middle portion (termed PC43) is the portion which interacts with huntingtin. Analysis of the primary structure of the HAP1 cDNA indicates three likely coiled coil structures, shown in Figure 1 B.


Figure 1. HAP1 constructs and probable coiled coil domains. (A) Schematic diagram of HAP1. The open reading frame is indicated as a box. The diagonal lines indicate the region of the cDNA identified in the screening with huntingtin (11) and the filled-in box indicates the approximate location of the presumed minimal huntingtin binding domain (HBD). The brackets indicate the four different constructs of HAP1 in the yeast two-hybrid vector: the N-terminus (amino acids 1-300), PC43 (amino acids 280-445) and the C-terminus of either HAP1-A (amino acids 421-599) or HAP1-B (amino acids 421-629). The helices represent the nucleotide position of the three putative coiled-coil structures of HAP1. (B) The probability that HAP1 forms coiled coils was estimated using the 28 amino acid window in the Coils program (27).

Four clones showed repeatable interaction with the middle portion of HAP1 (Table 1 and Fig. 2 A and B). These clones coded for proteins related to the cytoskeleton and included the p150Glued subunit of dynactin (18 -20 ), PCM-1 (21 ) and neurofilament M (22 ). In addition, we found a cDNA coding for a novel protein of ~0.7 kb with 68% homology to the human kinesin heavy chain (23 ). This clone comprises an [alpha]-helical stalk and part of the motor domain and probably corresponds to a conventional kinesin heavy chain isoform (data not shown). Two different clones for p150Glued were isolated with the yeast two-hybrid screen. For the other proteins only one independent clone was identified for each one. All of these cDNAs gave reproducible binding in the yeast two-hybrid system and the appropriate controls with either vector alone or c-Jun were negative (Fig. 2 A and B and data not shown). We also found interaction with two transcription factors, CREB-2 and KAP1/TIF1[beta] (data not shown) (24 -26 ). These clones are still under study and will be presented elsewhere. Like HAP1, all the clones have a high probability of forming coiled coils, as revealed by both the Coils (27 ) and Paircoil algorithms (28 ).


Figure 2. Association of HAP1 with itself and related cytoskeletal proteins. (A) [beta]-Galactosidase nitrocellulose filter lift assays showing that HAP1 interacts with p150Glued. Y190 were co-transformed with PC43 in pPC97 either with p150Glued in pPC86 or pPC86 alone. Co-transformation of c-Jun and c-Fos was used as a positive control for the [beta]-galactosidase assay. (B) Interaction of HAP1 with PCM-1, kinesin-like protein and neurofilament M. Y190 were co-transformed with PC43 in pPC97 and pPC86 containing either no insert or each of the interacting proteins. (C) HAP1 interacts with itself. Y190 were co-transformed with pPC97 and pPC86 containing either PC43 or the N-terminus of HAP1. As a control, pPC97, containing either PC43 or the N-terminus of HAP1, was co-transformed with pPC86 containing c-Jun.

During the library screening with the N-terminus and middle portion constructs it became apparent that these portions of HAP1 self-associate (data not shown). The N-terminus does not associate with the middle portion of HAP1 when co-transformed into yeast (Fig. 2 ). Neither of the C-termini of HAP1-A and HAP1-B associate with themselves nor with the other variant of the C-terminus (data not shown). The N-terminus and the middle portion of HAP1 (PC43) did not associate with c-Jun or c-Fos (Fig. 2 C and data not shown), which contain coiled coils (29 ).

Table 1 . HAP1 interacting proteins
  Interactor binding region
(amino acids)		 [beta]-Galactosidase activity Coiled coils
(Coils/Paircoil)		 GST-HAP1 binding
p150Glued subunit of dynactin 879-1150 +++ +/+ +
PCM-1 1279-1799 +++ +/+ +
Neurofilament M 6-407 ++ +/+ nd
Kinesin-like protein 359-583 +++ +/+ -
The amino acid positions of the clones that interact with HAP1 (PC43) are indicated relative to human p150Glued (accession no. X98801), PCM-1 (L27841), kinesin heavy chain cDNA (U06698) and rat neurofilament M cDNA (Z12152). [beta]-Galactosidase activity was strongly positive for p150Glued, PCM-1 and kinesin-related protein. All colonies turned blue after incubation for 10 min at 30°C. All clones isolated by yeast two-hybrid screening have a high probability of forming coiled coil secondary structure when analyzed by the programs Coils (http://ulrec3.unil.ch/software/COILS_form.html) and Paircoil (http://ostrich.lcs.mit.edu/cgi-bin/score).
nd, not determined.

In order to confirm the interactions at the protein level, in vitro binding assays were done using glutathione S-transferase (GST) fusion proteins linked to glutathione-Sepharose beads (Fig. 3 ). A fusion protein was constructed of GST ligated to the PC43 portion of HAP1 used as the probe in the yeast two-hybrid experiments. The interacting proteins were fused to a hemaglutinin (HA) epitope in the mammalian expression vector pRK5 and expressed in HEK 293 cells (30 ). Binding was detected with a monoclonal antibody against the HA epitope. In both cases there was consistent binding to the GST-HAP1 fusion protein, but not to GST alone (Fig. 3 A-E and Table 1 ). Binding was resistant to washing at high ionic strength and 0.5% Triton X-100. Under the conditions used in the experiment reproducible binding to the kinesin-related protein could not be demonstrated (data not shown).


Figure 3. In vitro binding of HAP1 fusion protein to P150Glued and PCM-1. (A) Coomassie blue staining of GST and GST-HAP1 (PC43) fusion proteins. (B and D) Western blots of lysates of HEK 293 cells expressing p150Glued-HA (amino acids 879-1150), PCM-1-HA (amino acids 1279-1799) or vector alone (control). (C and E) Binding of p150Glued-HA and PCM-1-HA to GST-HAP1 (PC43) but not to GST alone detected by Western blot.

Both HAP1 and the p150Glued subunit of dynactin are expressed at highest levels in the brain (11 ,16 ,31 ), so we performed immunoprecipitations from rat brain cytosol to look for an interaction between these two polypeptides in vivo. Immunoprecipitation experiments were performed from rat brain cytosol using an affinity-purified polyclonal antibody to p150Glued that has previously been demonstrated to co-immunoprecipitate dynactin subunits (20 ), as well as proteins which specifically interact with dynactin (32 ). The immunoprecipitates were resolved by SDS-PAGE and the corresponding immunoblots probed with an affinity-purified anti-peptide antibody raised against HAP1. As shown in Figure 4 , a polypeptide of ~100 kDa was observed in both rat brain cytosol and in the p150Glued immunoprecipitate. Under these conditions the antibodies depleted all p150Glued from the cytosol and about one third of HAP1. These immunoprecipitates were washed under stringent conditions, so this result suggests a strong in vivo association between p150Glued and HAP1. Neither HAP1 nor p150Glued were detected in controls performed in the absence of immunoprecipitating antibody (Fig. 4 ). We were not able to detect huntingtin in the p150Glued immunoprecipitate (data not shown).


Figure 4. HAP1 co-immunoprecipitates with dynactin. Dynactin was immunoprecipitated from rat brain cytosol using an affinity-purified antibody to p150Glued. Under these conditions complete depletion of dynactin from cytosol was observed. The resulting immunoprecipitates were resolved by electrophoresis (Coomassie stain for total protein shown in upper panel) and then immunoblotted with affinity-purified antibodies to p150Glued and to HAP1. Lane 1, cytosol prior to incubation with antibody-bound beads; lane 2, cytosol following immunodepletion; lane 3, proteins immunoprecipitated with affinity-purified antibody to p150Glued; lane 4, control precipitation with protein A-agarose beads. Control beads did not precipitate p150Glued or HAP1.


Figure 5. HAP1 and p150Glued are enriched in the same subcellular fraction. Protein fractions (25 µg/lane) were prepared as described in Materials and Methods, subjected to 4-15% gradient SDS-PAGE and analyzed by Western blot with monoclonal anti-p150Glued (1:1000) and polyclonal anti-HAP1 (AP157, 1:10 000) and anti-synapthophysin (1:3000) antibodies. All three proteins are enriched in the high speed pellet of the lysed crude synaptosomal fraction (LP2).

In order to study the subcellular distribution of p150Glued in relation to that of HAP1, we performed differential centrifugation experiments (Fig. 5 ). Under our homogenization conditions both HAP1 and p150Glued were highly enriched in the LP2 fraction. This fraction is enriched in synaptic and other vesicles (33 ). Gill et al. found that 10% of p150Glued is tightly bound to cytoskeleton components (19 ). We found p150Glued to be equally distributed in cytosolic and particulate fractions in brain, as has previously been shown (20 ). The LP2 fraction contains 0.7% of the protein content of the initial homogenate. HAP1 and p150Glued in this fraction accounts for 9.5 and 5.1% respectively of the original protein. This represents an enrichment of 14- and 7-fold in this fraction for HAP1 and p150Glued respectively. This was specific for the LP2 fraction and no enrichment was observed in any other fractions. The distribution patterns of HAP1 and p150Glued were similar to that observed for synaptophysin (Fig. 5 ) and synaptotagmin (data not shown), membrane proteins enriched in synaptic vesicles (34 ,35 ). These data are very similar to a previous observation for synaptophysin (34 ). HAP1 signal was detected in the crude homogenate after a longer exposure time (data not shown).

To determinewhether p150Glued and HAP1 are expressed in similar compartments within cells, we used immunofluorescence in PC12 cells treated with nerve growth factor (NGF) (Fig. 6 ; 36 ). Both HAP1 and p150Glued were most enriched in the perinuclear region. Some HAP1 labeling was also observed in the nucleus; however, the label was not abolished by preabsorption of the primary antibody with the immunogenic peptide, indicating non-specific staining in this region (data not shown). To further explore whether they co-localize within the cell, the sections were examined with a confocal microscope. There was a substantial overlap, as indicated by the yellow color, especially in this perinuclear region (Fig. 6 C).

DISCUSSION


Figure 6. Localization of HAP1 and p150Glued in PC12 neuronal cells. Confocal microscopy of PC12 cells differentiated with NGF and double labeled for HAP1 antibody (A, red) and anti-p150Glued antibody (B, green) as described under Materials and Methods. (C) Fused images of HAP1 (A) and p150Glued (B) showing that HAP1 and p150Glued co-localize, especially in the perinuclear region (in yellow). Bar 10 µm.In this study we have found that HAP1 binds specifically to the cytoskeletal proteins p150Glued and PCM-1. The interaction between HAP1 and the p150Glued subunit of dynactin was further examined by affinity chromatography and co-immunoprecipitation. The results from the two-hybrid screen, in vitro binding analysis and immunoprecipitation experiments all describe a specific interaction between these two proteins. These observations are consistent with previous observations that both HAP1 and p150Glued are most highly expressed in neurons (11 ,16 ,31 ) and that both these proteins are found in soluble and in vesicle-associated fractions (20 ). We have found that these two proteins also share similar subcellular localizations, as both are enriched in a nerve terminal vesicle fraction and partially overlapping distributions in PC12 cells were observed by immunocytochemistry.

HAP1 as well as the other proteins isolated by the yeast two-hybrid screen share a high probability of forming coiled coils. This observation suggests that the self-association of HAP1 which we observed, as well as the association of HAP1 with other cytoskeletal proteins such as p150Glued, may be mediated by formation of coiled coil interactions. However, the interactions we have identified appear to be specific. In control experiments we did not observe any association between HAP1 and c-Jun or c-Fos, which are known to dimerize via coiled coil interactions (29 ,37 ), and the HAP1 N-terminus did not associate with the HAP1 middle portion.

p150Glued is a subunit of dynactin, a complex formed from at least seven distinct subunits (19 ,32 ,38 , see 39 for a recent review). Dynactin binds both to microtubules and to the microtubule-based motor cytoplasmic dynein; both of these interactions are mediated by the p150Glued polypeptide (40 -42 ). The interaction between dynein and dynactin has been shown to be required for transport of vesicles along microtubules (19 ,43 ). In Drosophila the homolog of Glued is an essential gene; null mutants die early in embryogenesis (44 ). A dominant negative mutant in the Glued gene causes aberrant neuronal growth and morphology, most apparent in the compound eye and optic lobe (44 ,45 ).

Both dynein and dynactin are associated with vesicular organelles and are required for retrograde motility of these organelles along microtubules (19 ,20 ,43 ). This transport may be of particular importance in the extended processes of neuronal cells. Studies have also linked dynein and dynactin specifically to Golgi trafficking (32 ,46 ,47 ). It was shown that the amidating enzyme PAM (peptidylglycine [alpha]-amidating monooxygenase) binds to P-CIP10 (48 ,49 ). P-CIP10 is the rat homolog of human Duo cDNA, which we have found to associate with HAP1 (17 ). PAM is believed to have a role in biogenesis of large dense core vesicles (50 ).

It is notable that HAP1 can bind to regions of several different cytoskeletal proteins, all with predicted coiled coil domains. Leucine zipper coiled coil domains are important in the interactions of many transcription factors. Coiled coil interactions have also been implicated in interactions of many cytoskeletal proteins, including troponin T with dystrophin (51 ) and kinectin with kinesin (52 ,53 ). HAP1 associates in vitro with PCM-1, a protein potentially implicated in regulation of microtubule assembly (54 ). That HAP1 can potentially interact with several different cytoskeletal proteins, such as PCM-1, a kinesin-like polypeptide and neurofilament M, suggests that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins.

The localizations of HAP1 and p150Glued within cells fit well with previously published reports of the localization of huntingtin protein. Huntingtin has been found in association with several different vesicle populations (7 ,8 ). Huntingtin, like HAP1 and p150Glued, is enriched in the LP2 fraction (7 ). Huntingtin has also been localized by immunogold electron microscopy to axons in association with microtubules (10 ). A cytoskeletal localization of huntingtin is perhaps consistent with previous studies which have shown altered neuronal morphology in Golgi-labeled neurons in HD (55 ,56 ).

Huntingtin also interacts with huntingtin interacting protein 1, which shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae (14 ).

More recent studies of huntingtin (57 ,58 ) indicate that huntingtin is present in the trans-Golgi network and in several vesicle populations of the endocytic pathway leading to lysosomes. Huntingtin is also co-localized with the 58 kDa membrane-associated protein of the Golgi complex (58 ).

Therefore, these localizations of huntingtin and p150Glued are consistent with an indirect interaction via HAP1. This fits with previous hypotheses that one of the normal functions of the huntingtin protein may be in vesicle trafficking within cells (7 ,8 ,10 ). Furthermore, because the interaction between huntingtin and HAP1 is altered when the polyglutamine tract is expanded in HD patients, it is possible that these interactions may be involved in pathogenesis of the disease.

MATERIALS AND METHODS

Yeast two-hybrid constructs and screening

HAP1 subclones N-terminus, PC43, C-terminus of HAP1-A and C-terminus of HAP1-B (nucleotides 40-939, 877-1374, 1300-1836 and 1300-1926 respectively) were fused in-frame into yeast two-hybrid vectors containing the GAL4 DNA binding domain (pPC97) or activation domain (pPC86). C-Jun (amino acids 246-335) in pPC86 and c-Fos (amino acids 117-197) in pPC97 were used as controls for the yeast two-hybrid assays. The yeast strain Y190 (MATa, ura3-52, his3-[Delta]200, ade2-101, trp1-901, leu2-3, 112, gal4[Delta]gal80[Delta], URA::GAL-lacZ, cyhr2, LYS::GAL-HIS3) was used for all transformations. The yeast two-hybrid screening was performed as described previously (59 ,60 ).Briefly,a human fetal brain cDNA library in pPC86 was transformed into Y190 containing pPC97-HAP1 (PC43) (amino acids 280-445). The transformants were grown for 5 days in Trp-, Leu-, His- in the presence of 50 mM 3-amino-1,2,4-triazole (Sigma). The [beta]-galactosidase filter lift assays were performed for all grown colonies. The positive colonies were grown in Trp-, Leu-, His- medium and the plasmids rescued and transformed by electroporation into DH10B cells (Life Technologies). The cDNA clones in pPC86 were co-transformed with pPC97-HAP1 (PC43) to confirm the interactions.

Cell culture and transfection of HEK 293 cells

HEK 293 cells were grown in Dulbeco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 6 mM L-glutamine, 100 U/ml penicillin G (sodium sulfate) and 100 µg/ml streptomycin sulfate. Cells were transiently transfected by the calcium phosphate precipitation method (61 ) using 8 µg/10 cm plate plasmid DNA. p150Glued (amino acids 879-1150), PCM-1 (amino acids 1279-1799) or kinesin-related protein (amino acids 359-583) isolated from the yeast two-hybrid screen were inserted into pRK5 vector (Genentech), further modified with an HA tag. Cells were harvested 24-36 h after transfection.

Generation of HAP1 antibody (AP157)

A peptide (RQRSSMPAGSVTHC) corresponding to amino acids 401-413 of HAP1 plus a C-terminal cysteine (added for coupling purposes) was synthesized (Research Genetics) and conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) (Pierce) as described in the manufacturer's directions. New Zealand White rabbits were immunized with the conjugates (Cocalico Biologicals Inc., Reamstown, PA) using the KLH conjugate for the first two injections (days 0 and 14) and the BSA conjugate for subsequent boosts (days 21 and 51 and each 1-2 months thereafter). Antibodies were affinity purified from antisera collected after day 90. Antiserum (15-20 ml) was first treated by batch incubation overnight at 4°C with a Sulfolink column (Pierce) which had been previously reacted with 2-mercaptoethanol. Antibodies were then affinity purified using the peptide immunogen immobilized on a Sulfolink column essentially according to the directions of the manufacturer. The antiserum was incubated batchwise with 2 ml affinity matrix overnight at 4°C in the presence of 2 mM EDTA and a cocktail of protease inhibitors. The affinity resin was then poured into a column, washed extensively as described (7 ) and eluted with 90 mM glycine-HCl, pH 2.5, containing 10% ethylene glycol. The fractions collected were neutralized by addition of 100 µl 2 M Tris-HCl, pH 7.4. The fractions containing the highest absorbance at 280 nm were pooled and dialyzed extensively against 20 mM HEPES, pH 7.4, 0.9% NaCl followed by dialysis into 20 mM HEPES, pH 7.4, 100 mM NaCl and 50% glycerol. Antibodies were stored at -20°C. The anti-peptide antibody (AP157) recognized a band at the same location as the anti-fusion protein antibody (11 ) in a Western blot of protein extracts from transfected cells expressing HAP1-A.

Western blot analysis

Proteins were fractionated by 4-15% gradient SDS-PAGE and transferred to PVDF membranes (Schleicher & Schuell) at 400 mA for 2 h. The blots were blocked in 5% milk, phosphate-buffered saline, 0.1% Tween 20 for 1 h at room temperature and incubated with a 1:10 000 dilution of AP157, a 1:10 000 dilution of anti-HA (Babco), a 1:1000 dilution of anti-p150Glued (Transduction Laboratories) or a 1:3000 dilution of anti-synaptophysin (Boehringer Mannheim) for 1 h at room temperature. After washing in 5% milk/phosphate-buffered saline/0.1% Tween 20, blots were incubated with a 1:10 000 dilution of horseradish peroxidase-conjugated secondary antibody (Amersham) for 1 h at room temperature. Blots were developed using the enhanced chemiluminescence reagent (ECL kit, Amersham).

In vitro binding assay

The GST fusion protein of HAP1 was constructed by subcloning the middle portion of HAP1 (PC43) into pGEX-4T-2 (Pharmacia Biotech). Synthesis of recombinant proteins (pGEX-4T and pGEX-4T-2-HAP1) in BL21 cells (Novagen) was induced by 100 µM isopropyl-[beta]-D-thiogalactopyranoside for 2 h at 30°C. GST fusion proteins were purified on glutathione-Sepharose 4B beads (Pharmacia Biotech) according to the instructions of the manufacturer. For binding experiments HEK 293 cells transfected with p150Glued (amino acids 879-1150), PCM-1 (amino acids 1279-1799) or kinesin-related protein (amino acids 359-583) inserted into pRK5-HA were scraped off the plates and resuspended in 4 ml binding buffer containing 50 mM Tris, pH 7.4, 140 mM KCl and a protease inhibitor cocktail (Completetm; Boehringer Mannheim). Following sonication, 0.5% Triton X-100 was added and insoluble material was removed from the lysates by centrifugation at 12 000 g for 5 min. Supernatants of cell lysates were incubated with glutathione-Sepharose 4B beads containing GST or GST-HAP1 (PC43) fusion protein for 1 h at 4°C. Sepharose beads were washed three times with 1.5 ml phosphate-buffered saline and twice with 40 mM Tris, pH 7.4, 0.4 M NaCl and 0.5% Triton X-100. Proteins were eluted from beads with SDS sample buffer and detected by Western blot.

Immunoprecipitation

Two frozen rat brains were homogenized in PHEM buffer (50 mM sodium PIPES, 50 mM HEPES, 2 mM MgCl2 and 1 mM EDTA, pH 7.0) supplemented with tosylarginine methyl ester, pepstatin A, leupeptin, phenylmethylsulfonyl fluoride and 0.2 mM dithiothreitol, at a 1:1 (w/v) ratio. The homogenate was clarified by centrifugation at 39 000 g for 30 min at 4°C, followed by 60 min at 110 000 g. Cytosol was preabsorbed with protein A-agarose beads for 30 min at 4°C, then the beads were removed by centrifugation at low speed. Protein A-agarose beads which had been preincubated with affinity-purified anti-p150Glued antibody were then mixed with the cytosol and incubated for 3 h at 4°C. The antibody-bound beads were isolated by centrifugation at low speed, washed extensively and then eluted by boiling in gel sample buffer for SDS-PAGE. The immunoprecipitates were resolved on an 8% gel, then electroblotted onto Immobilon membrane (Millipore). After blocking the blots were probed with affinity-purified polyclonal antibodies to p150Glued (20 ), HAP1 or huntingtin (AP78) (7 ). The blots were developed with horseradish peroxidase-conjugated secondary antibodies and detected by chemoluminescence (ECL; Dupont NEN).

Subcellular fractionation

The subcellular fractionation was performed as described (7 ,33 ). Briefly, brains of 30 adult rats were homogenized in a buffer containing 0.32 M sucrose, 4 mM HEPES, pH 7.4, and a protease inhibitor cocktail (Completetm; Boehringer Mannheim). The crude homogenate (H) was centrifuged for 10 min at 800 g, producing the P1 pellet. The supernatant (S1) was centrifuged for 15 min at 9200 g, producing the P2 pellet and S2 supernatant. P2 was washed once with the original volume of homogenization buffer and centrifuged for 15 min at 10 000 g to produce the final P2 pellet. S2 supernatant was centrifuged for 2 h at 165 000 g to give the supernatant S3 and pellet P3. Washed P2 was resuspended in a small volume of homogenization buffer and lysed hypotonically with 9 vol. ice-cold water containing a cocktail of protease inhibitors. The pH was quickly brought to 7.4 by addition of concentrated HEPES, pH 7.4. The lysed P2 was centrifuged for 20 min at 25 000 g, giving rise to supernatant LS1 and pellet LP1. LS1 was centrifuged for 2 h at 165 000 g, producing supernatant LS2 and the crude synaptic vesicle fraction LP2. Protein samples (25 µg) were denatured for 5 min at 100°C in SDS sample buffer and monitored by Western blot analysis as described above. The relative intensity of the immunoblot bands was quantified using the program NIH image.

Immunocytochemistry

Rat pheochromacytoma cell line PC12 was differentiated to neurons by incubation with 10 µg/ml NGF for 4 days in serum-free medium (62 ). Cells were fixed with 4% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA and 3 mM MgCl2, pH 6.1) for 20 min at room temperature followed by 10 min in acetone plus 5 mM EGTA at -20°C. The cells were blocked in 5% normal donkey or goat serum containing 0.05% Triton X-100 for 30 min at 0°C and double labeled with AP157 (1.5 µg/ml) and anti-p150Glued (5 µg/ml) for 1 h at 37°C. Slides were incubated with 2 µg/ml Cy3-labeled donkey anti-rabbit and fluorescein-labeled donkey anti-mouse antibodies (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Slides were mounted using Vectashieldtm mounting medium (Vector Laboratories) and analyzed on a Noran confocal microscope at 100*.

GenBank accession number

The partial human kinesin-like protein cDNA sequence was deposited in the GenBank database (accession no. AF010146).

ACKNOWLEDGEMENTS

We thank K.Duan and C.Zhang for the technical support and Roxann Ashworth for DNA sequencing and analysis. This research was supported by a PEW fellowship award to S.E., a MSD award from the American Heart Association to V.C., a NIMH grant MH01152 to P.W. and NIH grant GM48661 to E.L.F.H. E.L.F.H. is an established investigator of The American Heart Association. The Baltimore HD Center is supported by NINDS grant NS16375, grants from the HDSA `Coalition for the Cure', bequests from Sar and Brita Ann Levitan and the DeVelbiss fund to C.A.R.

DISCLOSURE

Under an agreement between Guilford Pharmaceuticals Inc. and the Johns Hopkins University, the author is entitled to a share of sales royalty received by the University from Guilford Pharmaceuticals Inc. The terms of this arrangement are being managed by the University in accordance with its conflict of interest policies.

ABBREVIATIONS

BSA, bovine serum albumin; GST, glutathione S-transferase; HA, hemaglutinin; HD, Huntington's disease; HAP1, huntingtin-associated protein 1; KLH, keyhole limpet hemocyanin; NGF, nerve growth factor; PAM, peptidylglycine [alpha]-amidating monooxygenase.

REFERENCES

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NOTE ADDED IN PROOF

It was recently found that aggregates of an NH2-terminal fragment of mutant huntingtin are localized to dystrophic neurites and intranuclear inclusions in the HD brain [DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P. and Aronin, N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990-1993; Becher, M.W., Kotzuk, J.A., Davies, S.W., Bates, G.P., Price, D.L. and Ross, C.A. (1997) Intranuclear neuronal inclusions in Huntington's disease and dentatorubral and pallidoluysian atrophy: correlation between the density of inclusions and IT15 CAG triplet repeat length. Neurobiol. Dis., in press). Dysfunction in retrograde transport may lead to dystrophic neurites [DiFiglia et al. (1997) Science 277, 1990-1993; Sahenk, Z. and Lasek, R.J. (1988) Inhibition of proteolysis blocks anterograde-retrograde conversion of axonally transported vesicles. Brain Res. 460, 199-203], which is consistent with our proposal that HAP1 and huntingtin may play a role in retrograde vesicle trafficking, and that the mutation in huntingtin may alter this function.

*To whom correspondence should be addressed at: Ross 618, Johns Hopkins, 720 Rutland Avenue, Baltimore, MD 21205-2196, USA. Tel: +1 410 614 0011; Fax: +1 410 614 0013; Email: crgeppi@welchlink.welch.jhu.edu

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