HIP-I: A huntingtin interacting protein isolated by the yeast two-hybrid system
HIP-I: A huntingtin interacting protein isolated by the yeast two-hybrid systemErich E. Wanker*, Carlos Rovira, Eberhard Scherzinger, Renate Hasenbank, Stephanie Wälter, Danilo Tait, John Colicelli1 and Hans Lehrach
Max Planck Institut für Molekulare Genetik, Ihnestrasse 73, 14195 Berlin (Dahlem), Germany and 1Department of Biological Chemistry, UCLA School of Medicine and The Molecular Biology Institute, Los Angeles, CA90024, USA
Received November 7, 1996;Revised and Accepted January 2, 1997
We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments. For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential. The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein. Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis. The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks. The HIP1 gene is ubiquitously expressed in different brain regions at low level. HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain. HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.
Huntington's disease (HD) is an inherited neurodegenerative disorder which is characterized by uncontrolled movements, general motor impairments and dementia (1 ). The mutation responsible for HD has been identified as an expansion of an unstable CAG repeat located within the coding region of the HD (IT15) gene (2 ). The CAG repeat is highly polymorphic and varies from 6-34 repeats on chromosomes of unaffected individuals to 35-155 repeats on HD chromosomes (3 -5 ). The protein encoded by the IT15 gene, huntingtin, has a predicted molecular mass of ~350 kDa, depending on the number of glutamine residues. Both normal and mutated forms of huntingtin have been shown to be expressed at similar levels in the central nervous system and peripheral tissues (6 ,7 ). However, in HD there is a selective neuronal loss in distinct regions of the brain, with the caudate nucleus and putamen being the major sites of neuropathology, indicating that the pathology of HD is more likely to be due to a gain-of-function rather than to a loss-of-function. Several investigators have proposed that HD is due to a toxic gain-of-function caused by abnormal protein-protein interactions related to the elongated polyglutamine sequence (8 ). Thus, the binding of distinct proteins to the polyglutamine region could either confer a new property on huntingtin or alter its normal interactions with other proteins. It is possible that the specific binding of a protein with a restricted pattern of expression to the elongated polyglutamine stretch could cause selective vulnerability to particular cells.
To date, three potential huntingtin-interacting proteins have been identified. Using the two-hybrid system and a truncated huntingtin protein containing 44 glutamine repeats as a `bait', Li et al. identified a protein (HAP1) which is expressed predominantly in brain (9 ). The binding of HAP1 to huntingtin was shown to be enhanced by an expanded polyglutamine track. However, although HAP1 is expressed in both cortex and striatum, regions of the brain known to be affected in HD, it is also expressed in areas of the brain that are spared in HD. This suggests that factors other than HAP1 are causing vulnerability and selective neuronal death in HD. Burke et al. showed by biochemical methods that the glycolytic enzyme GAPDH binds to huntingtin and that this interaction is enhanced by an elongated polyglutamine track (10 ). The authors suggested that the binding of huntingtin to GAPDH might affect the energy metabolism in neuronal cells and that, ultimately, this could cause neuronal cell death by metabolic stress. However, the selective loss of neurons in the striatum cannot easily be explained with this hypothesis. Most recently, it was shown that the human ubiquitin conjugating enzyme hE2-25K, also named HIP2, binds selectively to the N-terminus of huntingtin (11 ). This protein is ubiquitously expressed in brain and its interaction with huntingtin does not appear to be modulated by the polyglutamine sequence. These results clearly show that huntingtin gets ubiquitinated, however, whether this has an effect on the pathology of HD or on the normal function of huntingtin is not known.
Despite the recent identification of three potential huntingtin interacting proteins, the normal cellular function of huntingtin is still unknown. Immunohistochemistry, electron microscopy and subcellular fractionations have shown that huntingtin is, primarily, a cytoplasmic protein associated with vesicles and/or microtubules (12 -14 ), suggesting that it may play a functional role in cytoskeletal anchoring or transport of mitochondria, vesicles or other organelles in the cell. However, besides the cytosol and membrane containing fractions, huntingtin has also been detected in the nucleus (15 ,16 ). Thus, a possible function in transcription regulation cannot be completely ruled out at this time, since the role of several polyglutamine-containing proteins in activating transcription is well documented (17 ).
To gain insight into the normal function of huntingtin and also to understand more about the pathology of HD due to the polyglutamine sequence, two-hybrid screens to isolate new huntingtin-interacting proteins were performed. We report here the discovery of a huntingtin-interacting protein, HIP-I, which exhibits significant sequence similarity to certain cytoskeleton proteins and which is expressed in various human tissues including brain.
To search for cDNA clones encoding proteins that interact with huntingtin, an improved version of the two-hybrid system was used. In this system, one hybrid is a fusion between the DNA-binding domain of the Escherichia coli LexA protein (amino acids 1-211) and a protein of interest, X, and the second hybrid is a fusion between the yeast GAL4 activation domain and some unknown protein, Y. Normally, these hybrids are unable to activate transcription by themselves, because one lacks a DNA-binding domain, and the other an activation domain. However, when both hybrids are coexpressed in Saccharomyces cerevisiae containing two integrated reporter constructs, the yeast HIS3 gene and the bacterial lacZ gene, which contain binding sites for the LexA protein, the interaction between X and Y leads to transcription of the reporter genes.To further improve the two-hybrid system the initial genetic selection (HIS3 and lacZ) (18 ) to isolate cDNAs encoding interacting proteins was coupled with an additional CAN-selection (19 ), resulting in the elimination of false positives. False positives are defined as library clones that activate transcription in cells expressing fusion proteins unrelated to the target protein. To eliminate these false positives, Trp+Leu+His+lacZ+ positive clones are grown in the presence of canavanine to select for loss of the plasmid containing the target fusion. The selection strain L40C is canR and the TRP1-LexA-DNA-binding domain fusion vector, pBTM117c (20 ), contains the wild-type CAN1 gene which confers canavanine sensitivity on a canR host. After selection on canavanine medium without leucine (SD-leu+CAN), the resulting strain is Trp-Leu+, maintains the LEU2 library plasmid and should be lacZ-, because the plasmid encoding the `bait' protein, with the LexA-DNA-binding domain, has been lost. This strain can now be used to isolate the LEU2 library plasmid and also can be retransformed with the original `bait' plasmid or plasmids expressing unrelated LexA-fusion proteins to determine the specificity of the protein-protein interactions.
As a `bait' for screening the human brain cDNA library the plasmid pBTM-HD1.7 (Fig. 1 ) was created. It contains a 1.7 kb IT-15 cDNA spanning amino acids 1-588 of huntingtin including a polyglutamine tract of 23 residues and one HEAT repeat (21 ). Because pBTM-HD1.7 alone lacked transcriptional activity, the yeast strain L40C containing this plasmid was transformed with a human brain cDNA library. A total of 8 million yeast transformants were placed under selection and 10 Leu+Trp+His+lacZ+ positive clones, which all were lacZ- on canavanine plates, were isolated. Of these yeast colonies, five stained intensively for [beta]-galactosidase activity within 1 h at 30oC. The library plasmids were isolated from these colonies and subsequent restriction analysis showed that each contained the same plasmid. One plasmid, carrying a 1.2 kb cDNA insert, was named pGAD-HIP1. The five other colonies isolated showed lacZ activity only following overnight incubation and are currently being characterized.
The deduced amino acid sequence encoded by the 1.2 kb HIP1 cDNA is shown in Figure 3 A. The principal open reading frame encodes a protein of 400 amino acids with a calculated molecular mass of 45 900 Da. The reading frame was confirmed by expressing the HIP1 cDNA in E.coli (Fig. 5 A). Since the cDNA sequence does not contain a potential translation initiation codon (26 ) or a stop codon, it was concluded that the fragment does not contain the entire coding region.
Database searches performed at the NCBI with the BLASTX program (27 ) showed no perfect matches to previously reported sequence data, indicating that HIP-I represents a novel protein. The highest score (61% similarity) was found between a 67 amino acid stretch close to the N-terminal end (positions 9-75) of HIP-I and a C.elegans hypothetical protein annotated ZK370 (Fig. 3 B) (28 ). The most similar sequence to this nematode ZK370 hypothetical protein is yeast Sla2p. Holtzman et al. (29 ) have shown that Sla2p is homologous to ZK370 and mouse talin (30 ), a protein that is believed to mediate cytoskeleton-membrane interactions. In comparison, Sla2p was shown to be essential for the assembly and function of the membrane cytoskeleton in S.cerevisiae. Additional database searches identified a periodic repetition of four leucine residues starting at position 291 using the ScanProsite tool searching for Prosite pattern entries. This pattern matches a leucine zipper with the consensus L-X(6)-L-X(6)-L-X(6)-L-X(6)-L (31 ). However, the functional relevance of this motif in HIP-I has to be determined by further biochemical studies.
To determine the size of the full length HIP1 cDNA, a Northern blot analysis of RNA from different brain tissues was performed (Fig. 4 A). Using the 1.2 kb HIP1 cDNA fragment as a probe, a single transcript with a size of ~8 kb was detected. HIP1 mRNA was present in all brain tissues examined, with the highest level found in the corpus callosum, hippocampus and substantia nigra. As the HIP1-probed blot in Figure 4 A was exposed for 10 times longer than the [beta]-actin-probed blot, HIP1 mRNA levels must be far lower than [beta]-actin mRNA in all tissues examined.
To further investigate the interaction between HIP-I and huntingtin, recombinant 6xHis-tagged HIP-I (HIP-His), glutathione S-transferase (GST) and GST-tagged N-terminal huntingtin (HD-GST) were produced in E.coli and purified to near homogeneity using affinity chromatography (32 ,33 ). Figure 5 A shows purified HIP-His, GST and HD-GST migrating on SDS-PAGE with apparent molecular masses of 55, 27 and 100 kDa, respectively. This is in good agreement with the predicted molecular masses based on the amino acid sequence.
For the in vitro binding reaction, recombinant HIP-His isolated from E.coli was incubated with immobilized HD-GST or immobilized GST and, following thorough washing, remaining bound proteins were separated by SDS-PAGE and immunoblotted with anti-HIP-I antibodies. Figure 5 B shows that the purified HIP-His protein was efficiently retained by the HD-GST protein linked to glutathione agarose beads, but a significant sticking of HIP-His was also detected with immobilized GST. A similar background level of bound HIP-His (20% of that seen with HD-GST) was also found with glutathione agarose beads without any immobilized protein (data not shown), and may represent some insoluble aggregates present in the HIP-His preparation. In a separate experiment recombinant HIP-His protein produced with the Baculovirus system was used for the in vitro binding reaction. Cleared lysates of Sf9 insect cells containing HIP-His were incubated either with immobilized HD-GST or immobilized GST. Figure 5 C shows that HIP-His was enriched from the protein extract by the E.coli HD-GST protein linked to glutathione agarose beads but not by the immobilized control protein GST. Taken together, these results indicate that recombinant HIP-I and huntingtin also bind to each other in vitro.
To determine whether HIP-I and huntingtin are localized in the same cellular compartments, subcellular fractions of human cortex were prepared by differential centrifugation (34 ). As shown in Figure 6 , HIP-I and huntingtin immunoreactivities were detected in both medium-speed (P2) and high-speed (P3) pellets, whereas the medium and high-speed supernatants (S2 and S3) exhibited little or no immunoreactivities. This result is in good agreement with those of DiFiglia et al. who demonstrated that huntingtin is enriched in compartments containing vesicle-associated proteins such as SV2, synaptophysin and the transferrin receptor (13 ). HIP-I and huntingtin behaved identically during subcellular fractionation, except for the crude nuclear fraction (P1). In this fraction, huntingtin was present at a low level, whereas HIP-I was present at a significantly higher level. These data suggest that huntingtin and HIP-I are associated with the vesicle compartment and, thus, have the possibility to come in physical contact with each other in the cell.
Using the two-hybrid system and an N-terminal huntingtin fragment as a `bait', we were able to isolate a potential huntingtin interacting protein (HIP-I). To examine the specificity of the observed in vivo interaction, the isolated library plasmid pGAP-HIP1 was retransformed into various yeast strains expressing different `bait' proteins (Fig. 2 A). We found that for a strong interaction to occur, HIP-I requires an N-terminal huntingtin segment of 588 amino acids. With a shorter N-terminal huntingtin protein (residues 1-171), which included the polyglutamine stretch, only a very weak interaction was detected, indicating that the region downstream of the polyglutamine sequence containing the HEAT repeat (21 ) is important for the interaction. HEAT repeats were found in different regulatory cytoplasmic proteins involved in cellular transport processes and they consist of two hydrophobic [alpha]-helices. Andrade and Bork (21 ) have proposed that HEAT repeats could be generally important for protein-protein interactions; however, whether the intact HEAT repeat in the huntingtin protein used to isolate HIP-I is essential for the interaction remains to be determined.
The C-terminal half of HIP-I contains a leucine zipper motif which could potentially mediate the interaction with huntingtin. However, our results with the two-hybrid system clearly show that the N-terminal half of HIP-I is sufficient for the interaction (Fig. 2 C), because deletion of the C-terminal domain, including the leucine zipper (Fig. 3 A), had no discernible effect on transcription activation. As there is no corresponding motif present in the N-terminal huntingtin protein used as a bait, the possibility that the HIP-I-huntingtin interaction occurs via a leucine zipper can be ruled out.
The possibility that an elongated polyglutamine sequence could influence the interaction between HIP-I and huntingtin was not addressed in this study. However, it is possible that such a sequence, which ultimately leads to the disease, could alter neighbouring domains in the native protein and, thereby, influence potential protein-protein interactions. Binding experiments with recombinant huntingtin proteins containing repeats of various lengths are currently in progress to test this hypothesis.
The in vivo results obtained with the two-hybrid system were confirmed with in vitro binding experiments. Figure 5 B and C show that recombinant HIP-His protein from both E.coli and Sf9 insect cells was selectively retained by HD-GST linked to glutathione agarose. However, additional experiments have to be performed to determine whether full length HIP-I and huntingtin are also associated in a native environment. Experiments to test whether HIP-I and huntingtin can be co-immunoprecipitated from brain extracts with a specific anti-HIP-I antibody and/or a specific anti-HD antibody are in progress.
A comparison of the HIP-I amino acid sequence encoded by the 1.2 kb cDNA with amino acid sequences in the NCBI databases using the BLASTX program revealed that HIP-I is related to the hypothetical 103.9 kDa ZK370 protein from C.elegans (28 ). The closest database hit to ZK370 was found to be yeast Sla2p, a cytoskeletal protein with a predicted size of 109 kDa. Holtzman et al. showed that especially the last 190 amino acids of Sla2p are highly homologous both to the C-terminus of ZK370 and the membrane associated mammalian protein talin (29 ,30 ). Thus, assuming that HIP-Iis related to ZK370 or Sla2p, it will be interesting to see whether a talin-related region is also present at the C-terminus of full length HIP-I protein.
Figure 4 shows that the HIP1 gene, similar to IT-15, is widely expressed in brain and peripheral tissues at a low level. Using the 1.2 kb HIP1 cDNA as a probe, a single mRNA species of ~8 kb was detected in all tissues examined. However, using an affinity-purified anti-HIP-I antibody, a single protein band migrating at ~116 kDa in SDS gels was identified in all cell extracts analysed (Fig. 4 B). This indicates that the HIP1 mRNA is much larger than the putative coding region for a 116 kDa protein. One explanation for the oversize of the HIP1 transcript is that it might contain a large 3' or 5' untranslated region (UTR). Unusually large 3'-UTRs were found in mRNAs for the synthesis of cytoskeleton proteins and it was proposed that these regions are important for targeting the mRNAs to particular regions in the cell (35 ). Whether this is also true for the HIP1 mRNA remains to be determined.
Our results demonstrate that HIP-I and huntingtin behave almost identically during subcellular fractionation and are enriched in the membrane containing fractions (Fig. 6 ). However, in the nuclear fraction there was a significantly higher level of HIP-I compared to huntingtin, indicating that huntingtin is selectively excluded from the nucleus. The finding that huntingtin is essentially absent from the nuclear fraction is in agreement with previous results obtained by cell fractionation experiments (12 ,13 ). However, this has to be investigated further, because preliminary experiments in our laboratory indicate that the absence of a signal in the nuclear fraction on Western blots could be due to aggregates of high molecular weight substances, which are mainly present in the nuclear fraction and which overlay the signal.
In general the results of the cell fractionation are in good agreement with previous biochemical and immunohistochemical evidence that huntingtin may contribute to protein trafficking or membrane cycling. Our data also support the hypothesis that huntingtin and HIP-I could be involved in the assembly and/or regulation of the cortical actin cytoskeleton underlying the plasma membrane, which is responsible for cell mobility, adhesion, membrane ruffling and transduction of extracellular signals to the interior of the cell (36 ). Although additional experiments are clearly necessary to test these hypotheses, we expect that a thorough analysis of the biological function of HIP-I will bring us a step further in understanding the normal function of huntingtin and also the pathogenesis of HD.
Saccharomyces cerevisiae strain L40C is isogenic with strain L40 (37 ) except for the presence of the can1 mutation. This mutation was selected by plating L40 cells on synthetic media without arginine and with canavanine (60 [mu]g/ml). The LexA DNA binding domain vector pBTM117c was generated from pBTM117 (20 ) by inserting an ~3.5 kb fragment of the CAN1 gene into the unique PvuII restriction site. The control plasmid pBTM-SIM1 (24 ) was a kind gift from M.R.Probst (UCLA, Los Angeles, CA). pBTM-HDXho, pBTM-HD1.7 and pBTM-HD3.6 were generated by ligating, respectively, 0.5 kb (nt 316-828), 1.7 kb (nt 316-2079) and 3.6 kb (nt 316-3954) IT-15 cDNAs (2 ) (as SalI-NotI fragments) into pBTM117c. For the construction of pBTM-MJD, a 0.6 kb MJD1 cDNA fragment (nt 496-1118) was obtained by PCR using plasmid HMJD1a (a generous gift from O. Riess, Ruhr-University, Bochum, Germany) and primers which contained either SalI or NotI sites at their 5'-ends. The amplified products were then digested with SalI and NotI and subcloned into pBTM117c. pGAD-HIPNT and pGAD-HIPCT were generated by ligating a 0.55 kb (nt 1-558) and a 0.65 kb (nt 559-1196) EcoRI HIP1 fragment, respectively, into pGAD10 (Clontech). The orientation of the inserts was determined by restriction analysis. All constructs were transformed into yeast L40C by the lithium acetate method (38 ) and the resulting transformants were tested for autoactivation of the lacZ and HIS3 marker genes. The open reading frames were verified by ABI dye terminator sequencing.For the construction of pAcHLT-HIP1, a 1.2 kb EcoRI HIP1 fragment was cloned into pAcHLT-A (PharMingen) and the orientation of the insert was determined by restriction analysis. Sf9 cells were co-transfected with pAcHLT-HIP1 and BaculoGoldTM DNA and recombinant viruses expressing the HIP1 gene were selected according to the manufacturer's recommendations (PharMingen).
A general description of the two-hybrid system has been detailed elsewhere (18 ). For our selection, a single colony of L40C cells, transformed with pBTM-HD1.7, was grown overnight in SD-trp medium (39 ) and transformed with a human adult brain cDNA library (MATCHMAKER, Clontech) constructed into plasmid pGAD10. A total of 8 * 106 independent transformants were plated on minimal medium lacking tryptophan, leucine and histidine. After incubation at 30oC for 5 days, colonies were picked, transferred into liquid SD-leu-trp-his medium and grown overnight to an OD600 of 1-2. Cells were then transferred into microtiter plates and spotted, in parallel, onto SD-leu-trp-his and SD-leu+CAN selective plates using a stamp. The plates were incubated at 30oC for 3 and 5 days, respectively. From both plates, replica filters were made and cells were permeabilized by freezing in liquid nitrogen (30 s) and thawing at room temperature. Filters were transferred onto Whatmann 3MM paper saturated with X-Gal solution (25 ) and incubated at 30oC. [beta]-Galactosidase positive clones on the filter from the SD-leu-trp-his plate, which were negative on the corresponding filter from the SD-leu+CAN plate, were used for further analysis. Single colonies from the SD-leu+CAN plates were grown overnight in liquid SD-leu medium. Total DNA was prepared from these cells and transformed into E.coli DH10B (BRL) by electroporation. Transformantswere selected on LB plates containing ampicillin. To check for true positives, isolated plasmids were transformed into L40C harbouring pBTM117c, pBTM-HDXho, pBTM-HD1.7, pBTM-HD3.6, pBTM-MJD or pBTM-SIM1 and tested for selective growth on SD-leu-trp-his plates and [beta]-galactosidase activity as before. The inserts of the library plasmids of positive clones were then sequenced.
In order to generate 6xHis-tagged HIP-I fusion protein (HIP-His), the HIP1 1.2 kb cDNA was released from pGAD10 by digestion with BamHI and EcoRI and subcloned into pBluescript II KS (Stratagene). From the resulting plasmid, pBKS-HIP1, a 1.2 kb BamHI-HindIII HIP1 fragment was isolated and ligated into the BamHI and HindIII site of pQE32N (Qiagen) creating pQE-HIP1. For the production of recombinant GAPDH-His protein, a 1.3 kb SalI-NotI GAPDH cDNA fragment (a gift from S.Meier-Ewert) was cloned in-frame into pQE30N (Qiagen) to generate pQE-GAPDH. pQE-HIP1 and pQE-GAPDH were transformed into E.coli SCS1 (Stratagene) carrying the lacIQ plasmid pREP4 (Qiagen) and protein expression was induced for 4 h with 1 mM IPTG. For the production of antibodies HIP-His and GAPDH-His were purified under denaturating conditions on a Ni-NTA affinity column according to the manufacturer's recommendations (Qiagen). For the purification of native HIP-His protein, induced cells were incubated in a buffer containing 40 mM Tris-HCl (pH 8), 0.1 M NaCl, 0.1 mM EDTA and 0.3 mg/ml lysozyme. After incubation for 45 min at 0oC, an equal volume of 20 mM Tris-HCl (pH 8), 0.3 M NaCl, 0.5% Brij58 was added, and the cell suspension was then gently stirred in a 37oC water bath until the temperature reached 20oC. The resulting lysate was centrifuged at 30 000 r.p.m. for 90 min in a Beckman 50Ti rotor and the supernatant was used for purification of HIP-His under native conditions according to the protocols of Qiagen. To produce GST-tagged N-terminal huntingtin (HD-GST), a 1.6 kb BamHI-NotI IT-15 cDNA fragment, was released from pSP-HD1.6[Delta] (E.E.Wanker and E.Scherzinger, in preparation) and subcloned into pGEX-5x-1 (Pharmacia). The resulting plasmid, pGEX-HD1.6[Delta], was transformed into E.coli DH10B (BRL) and protein expression was induced for 4 h with 1 mM IPTG. HD-GST protein was then affinity-purified using glutathione agarose beads (Sigma) and eluted with reduced glutathione as described (32 ).
Approximately 2.5 [mu]g of native HIP-His was incubated with 5 [mu]g GST or 3.7 [mu]g HD-GST immobilized on glutathione agarose beads in 0.2 ml HNTG buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol) for 12 h at 4oC. Beads were washed three times with high salt buffer (300 mM NaCl) to remove unbound HIP-His protein. Bound HIP-His was eluted from the beads with SDS sample buffer, separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane. Filters were incubated with affinity purified anti-HIP-I serum (1:100) followed by an anti-rabbit, secondary antibody conjugated to alkaline phosphatase.
For the in vitro binding experiments with the HIP-His protein produced in Sf9 cells, 50 [mu]l cleared lysate was added to 150 [mu]l HNTG buffer containing the affinity matrices described above. The mixture was rotated for 4 h at 4oC and after extensive washing of the beads with high salt buffer bound proteins were determined by Western blot analysis as described.
A commercially prepared blot loaded with mRNA from various human brain tissues (Clontech) was hybridized overnight with a randomly 32P-labeled 1.2 kb HIP1 fragment and a 2 kb [beta]-actin fragment, respectively. After washing of the blot according to the manufacturer's guidelines, the filter was exposed to X-ray film for 120 h for HIP1 and 12 h for [beta]-actin at -80oC.
To generate polyclonal antibodies against HIP-His and GAPDH-His the proteins, affinity-purified as described above, were injected into rabbits using standard immunisation procedures (40 ). The resulting immune sera were affinity-purified against the antigen immobilized on a Ni-NTA column as described (41 ).
For the identification of HIP-I in various tissues, whole cell extracts were prepared by homogenizing tissue samples (1 g) in 5 ml lysis buffer with protease inhibitors (10 mM Tris HCl pH 8, 2% Triton X-100, 1% SDS, 100 mM NaCl, 1 mM EDTA, 10 mM [beta]-mercaptoethanol, 2 mM PMSF, 10 [mu]g/ml leupeptin, 10 [mu]g/ml pepstatin, 1 [mu]g/ml aprotinin, 50 [mu]g/ml antipain) using 20 strokes of a glass homogenizer.
Subcellular fractions of human cortex were prepared essentially as described (34 ). Cortex was homogenized in 10 ml per gram wet weight of ice-cold 0.32 M sucrose in 4 mM HEPES (pH 7.4) containing 2 mM PMSF, 10 [mu]g/ml leupeptin, 10 [mu]g/ml pepstatin, 1 [mu]g/ml aprotinin, 50 [mu]g/ml antipain, using nine strokes of a glass homogenizer. The homogenate was centrifuged for 10 min at 1000 g to produce a pellet (P1), which was washed by resuspension in an equal volume of homogenization buffer and recentrifuged for 10 min at 1000 g. The original supernatant and wash were combined (S1) and then centrifuged at 17 500 g for 20 min yielding pellet (P2) and a supernatant (S2). The S2 fraction was centrifuged at 100 000 g for 60 min to give a high speed pellet (P3) and a high speed supernatant (S3). All steps were performed on ice. The protein fractions obtained by this method were separated on 6% SDS-gels and transferred to nitrocellulose membranes (42 ). Filters were incubated with antisera against HIP-I and huntingtin at dilutions of 1:100 and 1:500, respectively, followed by incubation with appropriate alkaline phosphatase conjugated secondary antibodies. For detection of huntingtin, the commercial monoclonal antibody 1HU-4C8 (Euromedex) was used.
For the preparation of insect cell lysates, Sf9 cells infected with viruses expressing HIP-His were lysed in 200 [mu]l of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 5 mM NaF, 5 mM NaPi, 5 mM NaPPi, 1 mM PMSF, 16 [mu]g/ml benzamidine, 10 [mu]g/ml phenanthroline, 10 [mu]g/ml aprotinin, 10 [mu]g/ml leupeptine and 10 [mu]g/ ml pepstatin A. After centrifugation at 14 000 g for 30 min cleared lysates were directly used for the in vitro binding experiments.
We thank O.Riess and M.Probst for supplying the plasmids HMJDa and pBTM-SIM1, respectively; G.Bruenning and S.Meier-Ewert for providing brain tissues and a GAPDH cDNA clone, respectively; G.Bates and S.Schnoegl for helpful comments and discussions; and D.Cahill for critical reading of the manuscript. The project was funded by the DFG grant `Analyse des menschlichen Genoms mit molekularbiologischen Methoden'.
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