| Human Molecular Genetics | Pages |
Ataxin-3 is transported into the nucleus and associates with the nuclear matrix
Introduction
Results
Characterization of anti-ataxin-3 antisera
Subcellular localization of the ataxin-3 protein by immunofluorescence and CLSM
Localization of ataxin-3 by biochemical subcellular fractionation
Discussion
Materials And Methods
Strains and plasmids
Purification of fusion proteins and antibody production
Cell lines and cell transfections
Immunofluorescence microscopy and CLSM
Western blot analysis
Cell fractionation and in situ matrix preparation
Acknowledgements
References
Ataxin-3 is transported into the nucleus and associates with the nuclear matrix
INTRODUCTION
Machado-Joseph disease (MJD), also called spinocerebellar ataxia type 3 (SCA3), is an inherited dominant autosomal neurodegenerative disorder with late onset of symptoms, generally after the 3rd or 4th decade. The manifestation of the disease includes cerebellar ataxia, pyramidal signs, nerve palsy, facial and lingual fasciculation and bulging eyes (1). In populations of Japanese origin, the disease affects ~1 in 106 persons (2). The mutation responsible for SCA3 has been identified as an expansion of an unstable CAG repeat located near the C-terminus of the SCA3 gene (MJD1) (3). The highly polymorphic SCA3 CAG repeat is normally 13-42 repeats in length (4), and is expanded to 61-84 repeat units in affected individuals. The SCA3gene is widely expressed in the central nervous system (5) and also in the peripheral tissues. It encodes a 42 kDa protein of unknown function. The SCA3 gene product, ataxin-3, was detected in human tissues as well as in transfected mammalian cells using anti-ataxin-3 antisera (6-8). Both the normal and mutated forms of ataxin-3 are expressed at similar levels in SCA3 lymphoblastoid cell lines (8). Immunohistochemistry of normal and diseased brain has revealed that the expression of the protein is restricted to a limited subset of neurons, particularly to those in the striatum (6). Furthermore, immunolocalization and biochemical studies performed with lymphoblastoid cells, transfected mammalian cells and with tissues of normal and diseased brains indicate that ataxin-3 is predominantly a cytoplasmic protein (6,8,9). In spite of the demonstration that an expanded polyglutamine sequence in the ataxin-3 protein leads to cell death (10), the physiological function of the normal protein remains unknown. Ikeda et al. (10) showed by immunoblot analysis that both the wild-type and mutant ataxin-3 proteins are present in the insoluble and soluble protein fractions prepared from transfected COS cells. The exact subcellular localization of the recombinant polyglutamine-containing proteins used in that study has not been determined. More recently, it was shown that the ataxin-3 protein with a polyglutamine sequence in the pathological range accumulates in ubiquitinated form in neuronal intranuclear inclusions (7), similar to those observed in Huntington's disease (HD) (11). Furthermore, there is evidence to indicate that an elongated polyglutamine sequence is necessary for the ataxin-3 protein to be transported into the nucleus, whereas the normal ataxin-3 protein was detected predominantly in the cytoplasm (6,7).
To gain insight into the normal function of ataxin-3, we undertook an immunocytochemical study to determine the exact subcellular localization of the protein in two different mammalian cell lines. Our results indicate that in both COS-7 and neuroblastoma cells the wild-type ataxin-3 protein is localized predominantly in the nucleus. This suggests that the protein, which contains a putative nuclear localization signal (NLS) close to the glutamine tract, per se has the ability to be transported into the nucleus.
RESULTS
Characterization of anti-ataxin-3 antisera
For the production of polyclonal antibodies against ataxin-3, two recombinant 6×His-tagged ataxin-3 fusion proteins were expressed in Escherichia coli and purified to near homogenity using immobilized metal chelate affinity chromatography (12). Figure 1a schematically shows the structure of the N-terminal (MJDNT-His) and C-terminal (MJDCT-His) fusion proteins corresponding to amino acids 1-178 and 179-360 of ataxin-3, respectively (3). For the generation of polyclonal antibodies, each of the recombinant fusion proteins was injected into rabbits and the resulting immune sera, NT1 and CT1, were affinity purified against the antigen immobilized on an Ni-NTA resin. To evaluate the specifity of the purified antibodies, the antisera were examined on western blots of E.coli cell extracts containing the recombinant full-length 50 kDa His-tagged ataxin-3 protein expressed from the plasmid pQE-MJD1a. Both antisera selectively recognized the relevant protein antigen on western blots (Fig. 1b). They failed to detect unrelated 6×His-tagged fusion proteins such as GAPDH-His (13) (data not shown) or any other protein present in the E.coli extracts. The specifity of both antibodies was also assessed using competition with specific and non-specific antigens, respectively (data not shown). The specificity of the NT1 antibody was assessed further by immunoblotting protein extracts prepared from human cortex and transfected or untransfected COS-7 cells. The COS-7 cells were transfected with a construct expressing the full-length ataxin-3 protein with a predicted Mr of 41.5 kDa (3). In the protein extract prepared from transfected COS-7 cells, a prominant band migrating at ~44 kDa was detected (Fig. 1c, lane 5). This band was not observed in the untransfected cells (Fig. 1c, lane 4). Furthermore, a higher molecular weight band migrating at ~50 kDa was observed in both the transfected and untransfected COS-7 cells and most likely represents the endogenous ataxin-3 protein (Fig. 1c, lanes 4 and 5). In the protein extract prepared from human cortex, besides the 50 kDa ataxin-3 protein, two additional proteins migrating at ~72 and 84 kDa were detected (Fig. 1c, lane 3). Whether these bands represent ataxin-3-related gene products or cross-reactive proteins is not known. It should be noted that similar higher molecular weight bands were also detected by Paulson et al. (6) using a different polyclonal anti-ataxin-3 antibody. The His-tagged ataxin-3 protein isolated from E.coli showed an Mr of ~50 kDa on western blots (Fig. 1c, lane 2). With the affinity-purified CT1 antibody, results similar to those shown in Figure 1c were obtained (data not shown).
| Figure 1. (a) Schematic representation of the ataxin-3 protein. The polyglutamine expansion (Q3KQ22) located near the C-terminus of the protein is represented by a black box. The regions responsible for nuclear targeting (CK-II and NLS) are represented by light boxes, and the amino acid sequence of this region is indicated above the schematic map. MJDNT-His and MJDCT-His indicate the portions of ataxin-3 used to generate antibodies. The numbers on the map indicate amino acid positions. (b)Western blot analysis of recombinant 6×His-tagged ataxin-3 protein expressed in E.coli and probed with NT1 and CT1 antisera. Whole cell extracts of E.coli prepared before (-) and after (+) induction of the ataxin-3 expression with IPTG were subjected to SDS-PAGE, blotted to nitrocellulose membranes and probed with the affinity-purified anti-ataxin-3 antibodies. (c) Detection of ataxin-3 in cell extracts prepared from transfected or untransfected COS-7 cells and in homogenates prepared from human brain cortex using the anti-ataxin antibody NT1. Lane 1, molecular mass standards; lane 2, purified His-tagged ataxin-3 protein (10 µg); lane 3, cell homogenate (20 µg) prepared from human cortex; lane 4, cell homogenate (20 µg) prepared from untransfected COS-7 cells; and lane 5, cell homogenate (20 µg) prepared from COS-7 cells transfected with the plasmid pSG5-MJD. Arrows on the right point to the ataxin-3 bands migrating at ~44 and 50 kDa, respectively. |
|
Subcellular localization of the ataxin-3 protein by immunofluorescence and CLSM
The subcellular localization of ataxin-3 in untransfected and transiently transfected COS-7 cells expressing the full-length ataxin-3 protein with a polyglutamine sequence in the normal range (Q3KQ22) was analysed by immunofluorescence microscopy (Fig. 2). The cells were fixed using paraformaldehyde as well as two alternative methods (methanol and methanol/acetone 1:1; data not shown), permeabilized and probed with the anti-ataxin-3 antibodies NT1 or CT1, followed by a Cy3- conjugated anti-rabbit antiserum. In the transfected cells, a strong nuclear and a strong cytoplasmic signal was detected with both antibodies (Fig. 2A and B). In comparison, in the untransfected COS-7 cells, we observed that the nuclear staining was much stronger than the cytoplasmic staining (Fig. 2C). In contrast, the affinity-purified anti-GAPDH control serum (13) showed only a diffuse cytoplasmic staining (Fig. 2E), as expected. A strong nuclear and significantly weaker cytoplasmic staining was also observed when the neuroblastoma cell line SHSY5Y was examined by immunofluorescence microscopy using the NT1 antibody (data not shown).
Figure 2. Immunofluorescence analysis of transfected and untransfected COS-7 cells. Transfected COS-7 cells expressing endogenous and recombinant full-length ataxin-3 were probed with the anti-ataxin-3 antibodies NT1 (A) or CT1 (B). Untransfected COS-7 cells were immunolabelled with the anti-ataxin-3 antibody NT1 (C) or with the anti-GAPDH antibody (D). To examine the subcellular distribution of the ataxin-3 protein in the neuroblastoma cell line SHSY5Y in more detail, whole cell monolayers (Fig. 3A and B), in situ matrix preparations (Fig. 3C and D) and isolated nuclei (Fig. 3E and F) were examined by confocal laser scanning microscopy (CLSM). Using antibodies NT1 or CT1 in the neuroblastoma cells (Fig. 3A and B), a strong fluorescent signal in the nuclei, particularly in the nucleoplasmic regions, was detected, whereas it was almost absent from the cytoplasm. The nuclear fluorescence could be observed in different serial optical sections and was totally removed by pre-incubating the antisera with the specific antigen (data not shown). A fluorescent staining with about the same intensity was also obtained when in situ matrix preparations or purified nuclei were examined by CLSM (Fig. 3C-F). Using the antisera NT1 or CT1 in the in situ matrix preparations and in the purified nuclei, an immunofluorescence signal was detected in the inner nuclear matrix area and the nucleoplasmic regions, respectively. In contrast, only a very weak signal was detected in the nucleolar remnants (Fig. 3C and D), indicating that the ataxin-3 protein present in the nuclei of neuroblastoma cells is associated primarily with the nuclear scaffold (14,15). When the neuroblastoma cell line IMR-32 was probed with the antibodies NT1 or CT1, results similar to those shown in Figure 3 for the SHSY5Y cells were obtained (data not shown). Figure 3. Analysis of the subcellular localization of ataxin-3 using confocal laser scanning microscopy (CLSM). Whole SHSY5Y neuroblastoma cells (A and B), in situ matrix preparation (C and D) and isolated nuclei (E and F) were probed with the anti-ataxin-3 antibodies NT1 (A, C and E) or CT1 (B, D and F). Images generated with the confocal microscope were analysed as described.
Localization of ataxin-3 by biochemical subcellular fractionation
To determine the subcellular localization of ataxin-3 in transfected COS-7 and untransfected neuroblastoma cells, nuclear (N) and cytoplasmic (C) protein fractions were prepared by centrifugation (16). The protein fractions were separated by SDS-PAGE and analysed by western blotting using the affinity-purified antibody NT1. Figure 4a shows that in both nuclear and cytoplasmic protein fractions prepared from the transfected COS-7 cells about the same amount of ataxin-3 immunoreactivity was present. In comparison, in the nuclear fraction prepared from the neuroblastoma cells SHSY5Y, the immunoreactive band was clearly more pronounced than in the cytoplasmic fraction (Fig. 4b). This result is in good agreement with those obtained by immunofluorescence microscopy and CLSM (Figs 2 and 3). As a control for the purity of the cellular fractions, proteins with a well-known subcellular distribution were also tested on the same western blot. Figure 4a and b shows that the cytoplasmic protein GAPDH was only present in the cytoplasmic fraction, whereas the histone proteins were detected exclusively in the nuclear fraction, as expected.
| Figure 4. Subcellular fractionation of cell extracts prepared from transfected COS-7 (a) or untransfected SHSY5Y neuroblastoma cells (b). Protein fractions were prepared according to the method of Sittler et al. (15), resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with the anti-ataxin-3 antibody NT1 (upper panel), the anti-GAPDH antibody (middle panel) or the anti-histone antibody (lower panel). T, whole cell extract; C, cytoplasmic fraction; N, nuclear fraction. |  |
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DISCUSSION
Previous immunohistochemical studies of human brain have localized the ataxin-3 protein containing a polyglutamine sequence in the normal range (13-42Q) to both neuronal and non-neuronal cells in a predominantly cytoplasmic distribution. In contrast, in SCA3 diseased brains, the protein was detected primarily in the nuclei of neurons (6,7), suggesting that an expanded glutamine repeat is responsible for the translocation of ataxin-3 into the nucleus. In the present work, to determine whether a full-length ataxin-3 protein with a glutamine repeat in the normal range (Q3KQ22) is transported into the nucleus, COS-7 as well as neuroblastoma cells were examined by immunofluorescence microscopy, CLSM and subcellular fractionations. Using the purified anti-ataxin-3 antibodies NT1 or CT1, the ataxin-3 protein was detected predominantly in the nucleus of both untransfected mammalian cells (Figs 2C and 3A and B), indicating that an elongated glutamine sequence is not necessary for the protein to be transported into the nucleus. In comparison, in COS-7 cells that have been transfected, a nuclear as well as a cytoplasmic staining was detected, which is in agreement with the results obtained by Paulson et al. (7) who found for kidney epithelial 293T cells that the expression of the full-length ataxin-3 protein with a normal (27Q) or an expanded glutamine tract (78Q) resulted in a diffuse cytoplasmic staining. On the other hand, a truncated ataxin-3 protein with an expanded glutamine repeat did not distribute diffusely within the cytoplasm of the 293T cells, but rather localized to distinct subcellular structures in the cytoplasm and also in the nucleus, indicating that it is mainly the truncated ataxin-3 protein with an elongated glutamine sequence which translocates to the nucleus, where it eventually forms microaggregates (7). In addition, evidence was presented that full-length ataxin-3 protein with a glutamine repeat in the normal range is recruited by the truncated form of ataxin-3 into the nuclear aggregates. However, our results obtained with the untransfected COS-7 and neuroblastoma cells indicate that neither a truncation nor an expanded glutamine sequence is a prerequisite for the nuclear transport.
It was shown recently for ataxin-1 that both the wild-type and mutated protein, containing glutamine repeats in the normal or pathological range, respectively, are localized in the nuclei of cerebellar Purkinje cells or transfected COS-1 cells, indicating that this disease protein is transported into the nucleus independently of the presence of an elongated glutamine repeat (17). Furthermore, it was demonstrated that both the normal and the mutated ataxin-1 protein associate with the nuclear matrix. In our study, a strong fluorescent staining was detected when in situ matrix preparations or purified nuclei of neuroblastoma cells were examined by CLSM (Fig. 3C-F), indicating that ataxin-3, similarly to ataxin-1, is associated with the nuclear matrix. It was proposed by Skinner et al. (17) that a critical aspect of SCA1 pathogenesis could be the disruption of a nuclear matrix-associated protein domain because, unlike wild-type ataxin-1 which localizes to several small nuclear structures, the mutated protein localizes to a single, defined 2 µm structure before the onset of ataxia. Although additional experiments will be necessary to ascertain that the aggregated ataxin-3 protein present in the nuclear inclusions in SCA3 brains is in fact associated with the nuclear matrix and causes alterations of the normal nuclear structure, we suggest that also in this disease a disruption of a matrix-associated protein domain could be responsible for the pathogenesis. As neuronal nuclear inclusions containing huntingtin aggregates have been detected in transgenic mice expressing a fragment (exon 1) of the HD gene (18,19) as well as in the brain of HD patients (11), in the future the nuclear distribution of huntingtin needs to be analysed in more detail in HD patients.
To explore whether the normal ataxin-3 contains an NLS, database searches were performed using the PSORT program (20,21). Figure 1a shows that there is a potential NLS as well as two casein kinase II (CK-II) sites immediately upstream of the polyglutamine sequence. It has been demonstrated that proteins that harbor an NLS very often also contain CK-II sites at a distance of ~10-30 amino acid residues from the NLS (22). While the NLS determines the specificity of the nuclear transport, the CK-II site determines the rate of the transport. Additional experiments will be necessary to determine whether the potential NLS and CK-II sites in ataxin-3 are essential for the translocation of the protein into the nucleus. Interestingly, a putative NLS and two CK-II sites very similar to the motifs present in the ataxin-3 protein were also detected in ataxin-1 (data not shown), a protein known to translocate into the nucleus (17).
Taken together, our results suggest that both ataxin-3 and ataxin-1 are transported into the nucleus of mammalian cells by a similar mechanism and that the protein which enters the nucleus associates with the nuclear matrix. Furthermore, our immunocytochemical studies provide the basis to unravel the precise functional role of ataxin-3 in the nucleus of mammalian cells.
MATERIALS AND METHODS
Strains and plasmids
Escherichia coli DH10B was used as the host for the construction of expression plasmids. For the production of the recombinant proteins, the plasmids were transformed into E.coli SCS1 (Stratagene) carrying the lacIQ plasmid pREP4 (Qiagen). pQE-MJD1a was generated by subcloning a 1.1 kb BamHI-NotI SCA3 cDNA fragment into pQE32N, a derivative of pQE32 (Qiagen) containing BglII and NotI sites in place of the PstI multilinker site. The 1.1 kb SCA3 cDNA fragment was obtained by PCR using plasmid HMJD1a (a generous gift from O. Riess, Ruhr-University, Bochum, Germany) and the primers 5[prime]-ATAGTTTAGCGGCCGCTTATGTCAGATAAAGTGTGAAGGT and 5[prime]-CGGGATCCAGTCGACGATGGAGTCCATCTTCCACG which contain either BamHI or NotI sites on their 5[prime] ends. For the construction of pQE-MJDCT, a 0.6 kb SalI-NotI SCA3 cDNA PCR fragment (13) was ligated into SalI-NotI-digested pQE32N. pQE-MJDNT was created by deleting a 0.5 kb PstI-HindIII SCA3 cDNAfragment from pQE-MJD1a and religating the vector after filling-in the ends with T4 DNA polymerase. pSG5-MJD was generated by ligating a 1.1 kb BamHI SCA3 cDNA fragment, isolated from pQE-MJD1a, into the BglII restriction site of pSG5 (23). The orientation of the insert in pSG5-MJD was verified by restriction analysis.
Purification of fusion proteins and antibody production
In order to generate the 6×His-tagged ataxin-3 fusion proteins MJDNT-His and MJDCT-His, the expression plasmids pQE-MJDNT and pQE-MJDCT, respectively, were transformed into E.coli SCS1 (pREP4). Expression of the recombinant proteins was induced for 4 h with 1 mM isopropyl-[beta]-d-thiogalactopyranoside (IPTG) and the His-tagged proteins were purified under denaturating conditions on an Ni-NTA column as described (12). For the generation of antibodies against MJDNT-His and MJDCT-His, the purified protein fractions were injected into rabbits (24) and the resulting immune sera were affinity purified against the antigen immobilized on an Ni-NTA column (25).
Cell lines and cell transfections
COS-7 cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 5% fetal calf serum (FCS) and containing penicillin (5 U/ml) and streptomycin (5 µg/ml). The human neuroblastoma cells lines IMR-32 and SHSY5Y (26) were maintained in the same medium supplemented with 12% FCS. For the transient expression of the ataxin-3 protein, COS-7 cells were plated to 30% confluence in 9 cm dishes and transfected with 10 µg of pSG5-MJD DNA and 10 µg of carrier DNA by the calcium phosphate co-precipitation technique (27). Forty hours after transfection, the cells were harvested and cell pellets were homogenized in lysis buffer with protease inhibitors [50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 µg/ml aprotinin, 50 µg/ml antipain]. After centrifugation, the cleared lysates were used for western blot analysis.
Immunofluorescence microscopy and CLSM
Neuroblastoma cells and transfected and untransfected COS-7 cells were grown in Leighton tubes (Costar) and prepared for immunofluorescence detection as described (16). After fixation in 2% paraformaldehyde for 4 min at room temperature, the cells were permeabilized with 0.1% Triton X-100 for 15 min and washed extensively with phosphate-buffered saline (PBS). The cells were then incubated with the antibodies NT1 or CT1 at dilutions of 1:200 and 1:100, respectively, followed by incubation with the conjugated Cy3 donkey anti-rabbit antibody (Jackson Laboratories). After extensive washing with PBS and 0.1% Triton X-100, the nuclei were counterstained with Hoechst (Sigma). The samples were observed with the fluorescence microscope Axiophot-2 (Zeiss). Two alternative methods of fixation (methanol and methanol/acetone 1:1) were tested to confirm the results obtained with the paraformaldehyde method.
For the subcellular localization of the ataxin-3 protein in neuroblastoma cells by CLSM, whole cells, in situ matrix preparations or isolated nuclei were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (28) and incubated with the antibodies NT1 or CT1 at dilutions of 1:100 and 1:10, respectively. The secondary antibody used was a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (Dako). The observations were carried out using a Sarastro Phoibos 1000 confocal microscope (Molecular Dynamics, Sunnyvale, CA). In brief, for image acquisition, the samples were excited with the 488 nm line of the argon-ion laser attenuated at 10% with the neutral density filter. The emission signal was observed through a dichroic mirror (500 nm) followed by a long pass filter (530 nm). The spatial projections were reconstructed by using ImageSpace software (Molecular Dynamics) running on a workstation Indigo (Silicon Graphics, Mountain View, CA).
Western blot analysis
For immunoblots, the proteins present in the nuclear, cytoplasmic and whole cell extracts were if necessary concentrated by trichloroacetic acid (TCA) precipitation (28) and separated by SDS-PAGE (29). Proteins were electrophoretically transferred to nitrocellulose (30). The membranes were blocked in 3% non-fat dry milk and incubated with the antibodies NT1, CT1, anti-GAPDH and anti-histone at dilutions of 1:10 000, 1:1000, 1:5000 and 1:5000 respectively, followed by an anti-rabbit or anti-mouse peroxidase-conjugated antibody (Sigma). The blots were developed with the ECL kit (Amersham). Protein concentration was determined by the Bio-Rad assay.
Cell fractionation and in situ matrix preparation
Nuclear (N) and cytoplasmic (C) protein fractions from COS-7 cells were prepared essentially as described by Sittler et al. (16). Highly pure nuclei of neuroblastoma cells were obtained by hypotonic shock in combination with a non-ionic detergent extraction method (31). For preparation of the cytoplasmic fraction, the nuclear pellet was removed by centrifugation and the supernatant was recentrifuged to avoid contamination by nuclear debris.
The in situ matrix preparation was carried out as described (28). In brief, adherent cells were permeabilized for 10 min at room temperature with TSM buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 150 mM NaCl) containing 1% NP-40 and 2 mM sodium tetrathionate. After washing in TSM buffer, the samples were incubated with 30 U/ml DNase I (Sigma, St Louis, MO) in TSM for 1 h at room temperature. After washing in TSM, the cells were extracted twice with 2 M NaCl in TSM for 5 min each. Finally, the samples were washed in PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 4°C.
ACKNOWLEDGEMENTS
We thank S. Schnoegl for reading the manuscript, A. Valmori for developing the photographs, J. Wirth for helping with the fluorescent microscope, and J. Priller for SCA3 lymphoblastoid cells. The project was funded by the DFG grant `Funktionelle Analyse von Huntingtin und MJD1'.
REFERENCES
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Y. Chai, L. Wu, J. D. Griffin, and H. L. Paulson
The Role of Protein Composition in Specifying Nuclear Inclusion Formation in Polyglutamine Disease
J. Biol. Chem.,
November 21, 2001;
276(48):
44889 - 44897.
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