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Supplemental Data
1. Experimental procedures and materials
Plasmid constructions
The plasmids pGEX-6P-Atx22Q(2-360), pGEX-6P-Atx22Q(221-360), pGEX-6P-Atx(2-220),
pGEX-6P-Atx(2-256), pGEX-6P-Atx(221-291) and pGEX-6P-Atx(257-291) were generated
by amplifying MJD1 cDNA-fragments from pQE-MJD1a (Tait et al., 1998) and subcloning
them into pGEX-6P-1 (Amersham Biosciences, Freiburg, Germany). pGEX-6P-Atx70Q(2360), pGEX-6P-Atx71Q(242-360), pGEX-6P-Atx71Q(257-360), pGEX-6P-Atx43Q(2-360),
pGEX-6P-Atx71Q(186-360) and pGEX-6P-Atx46Q(221-324) were constructed by
amplifying MJD1 cDNA fragments from pBSK-MJD74Q (a gift from F. Laccone) and
subcloning them into pGEX-6P-1. pGEX-6P-Atx71Q(257-360)282HNHH, pGEX-6PAtx68Q(242-360)282HNHH and pGEX-6P-Atx68Q(2-360)282HNHH were generated from
pGEX-6P-Atx71Q(257-360) or pGEX-6P-Atx70Q(2-360) by site directed mutagenesis using
the Quik Change Kit from Stratagene. The plasmid pGEX-6P-Atx22Q(2-360)282HNHH was
obtained by site directed mutagenesis from pGEX-6P-Atx22Q(2-360). pGEX-6PAtx27Q(281-360) was generated by subcloning the cDNA fragment from MJDtr-Q27
(Warrick et al., 1998) to pGEX-6P-1. Plasmids pGEX-5X-Htt51Q, pGEX-5X-Htt20Q and
pGEX-5X-HIP1(218-604) have been described (Scherzinger et al., 1999; Waelter et al.,
2001). To generate pTL-Atx22Q and pTL-Atx70Q, MJD1 cDNA fragments were amplified
from pGEX-6P-Atx22Q and pGEX-6P-Atx70Q, and subcloned into pTL1 (Kastner et al.,
1992). Plasmids pcDNAHtt17Q(1-510) and pcDNAHtt68Q(1-510) were generated by
insertion of fragments coding for Htt17Q(1-510) and Htt68Q(1-510) into pcDNA-I (Life
Technologies, Gaithersburg, MD). The cDNA encoding VCP was obtained from ATCC
(American Type Culture Collection, Manassas), amplified, and cloned into pTL-Flag10 (a gift
from D. Devys and J.-L. Mandel). To produce a His-tagged VCP fusion protein, the VCP
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cDNA was amplified by PCR and subcloned into pQE31N, a derivative of pQE31 (Qiagen,
Hilden, Germany). pUAS-VCP was generated by subcloning the VCP cDNA into the pUAST
transformation vector. The cDNA encoding Ufd1 was obtained from the RZPD (Deutsches
Ressourcenzentrum für Genomforschung GmbH), amplified, and cloned into pGEX-6P-1 and
pMAL-c2X (New England BioLabs, Frankfurt am Main, Germany).
pGEX-6P-E4B(2-600) was generated by amplifying a cDNA fragment from pEF-DEST51E4B (a gift from J.A. Mahoney) and subcloning into pGEX-6P-1. The plasmid pGEX-4T-synuclein (A53T) was a gift from C.A. Ross. All constructs were verified by DNA
sequencing.
Protein expression and in vitro binding experiments
GST-, MBP- and His-tagged fusion proteins were expressed in E. coli and purified on affinity
columns as previously described (Scherzinger et al., 1997) and according to manufacturer’s
instructions (New England Biolabs, Frankfurt am Main, Germany; Qiagen, Hilden, Germany).
For in vitro binding experiments, GST fusion proteins (2-10 µM) were incubated with human
brain extract (2.65 mg/ml), reticulocyte lysate (50 mg/ml) or purified His-VCP (0.5 µM
hexamer) in binding buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM
DTT, 1 mg/ml ovalbumin) in the presence of 2 mM nucleotide or 20 U/ml apyrase (Sigma, St.
Louis; MO) at 30°C for 30 min. Then, glutathione agarose beads were added, and incubation
continued at 4°C for 1 h with rotation. Beads were washed 5 times with binding buffer
(without ovalbumin) containing 0.1% NP-40. Bound proteins were eluted from the beads with
SDS-sample buffer, boiled for 5 min and analysed by immunoblotting.
Competition experiments were carried out as described above using 0.1 to 1 mM BP43 (AcTSEELRKRREAYFEK-NH2), BP-E4B (Ac-SADEIRRRRLARLAG- NH2) or SP43 (AcRYTEFERSKEKRALE- NH2) in the binding reaction.
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Mass spectrometry
For protein identification, a 100 kDa band was excised from a 1D gel and in-gel digested with
trypsin. After chromatographic separation of the peptide mixture on a 75 µm PepMap C18
column (Dionex, Idstein, Germany), the peptides were identified by a capillary liquid
chromatography system delivering a gradient of 5 to 50% acetonitrile. The eluting peptides
were then ionized by electrospray ionization (ESI) on a Q-TOF hybrid mass spectrometer
(Micromass, Manchester, UK). The MS/MS analyses were conducted by using collision
energy profiles, which were chosen based on the m/z value, charge state of the parent ion,
fragment ion masses and intensities, and correlated with the protein database MSDB using
Mascot software (Perkins et al., 1999).
Antibodies
To generate a polyclonal antibody against His-VCP, the recombinant protein was affinitypurified and injected into rabbits using standard immunisation procedures. The resulting
immune serum was affinity purified against the antigen immobilized on a Ni-NTA column.
The polyclonal anti-Atx-3 antibody, CT1, and anti-Htt antibody, HD1, have been described
previously (Scherzinger et al., 1997; Tait et al., 1998). Commercially available antibodies are:
Anti-Flag (Sigma, St. Louis, MO), anti-Ufd1 (Transduction Laboratories, Lexington, KY),
anti-GST (Amersham Biosciences, Freiburg, Germany), anti-dynamin (Transduction
Laboratories, Lexington, KY), anti-Htt, 4C8 (Chemicon, Temecula, CA) and anti-Atx-3, 1H9
(Chemicon, Temecula, CA).
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Microscopic Analysis
For immunofluorescence microscopy, formalin fixed SCA3 pons sections (Evert et al., 2003)
were processed and labeled with mouse monoclonal, 1H9 antibody (1:1500) and anti-VCP
antibody (1:1000), followed by Texas Red goat anti-mouse and fluorescein 5-(4,6dichlorotriazin-2-ylamino) fluorescein goat anti-rabbit antibodies (Jackson
ImmunoResearch, West Grove, PA). Samples were viewed with a Nikon Eclipse E800
fluorescence microscope (Nikon, Düsseldorf, Germany). Digitized images were collected on
separate fluorescence channels using a Sony 3CCD digital camera.
Bioinformatics analysis
Protein sequences were retrieved from the NCBI database (Wheeler et al., 2005). The
multiple sequence alignment of Atx-3 homologs (Fig. 4E) was computed by the MUSCLE
program (Edgar, 2004) and edited using SEAVIEW (Galtier et al., 1996). The sequence logo
was drawn using WebLogo (Crooks et al., 2004). Potential nuclear localization signals were
detected using the online prediction service PSORT II (Nakai and Horton, 1999) .
Cell transfections and immunoprecipitations
COS-1 cells were grown in Dulbecco’s modified Eagle medium (Life Technologies,
Gaithersburg, MD) supplemented with 5 % fetal calf serum, penicillin (100 IU/ml) and
streptomycin (100 µg/ml). Transfections of pTL-Atx22Q, pTL-Atx70Q, pcDNAHtt17Q(1510), pcDNAHtt68Q(1-510) and pTL-FlagVCP were performed with Lipofectamine Plus
(Life Technologies, Gaithersburg, MD).
For co-immunoprecipitations, cells were harvested 40 h post transfection, lysed, and
processed, as previously described (Sittler et al., 1998). For each immunoprecipitation
experiment, 200 µg protein was incubated with the respective antibody (0.5 µl CT1 or 1.3 µl
HD1) in 200 µl NP-40 lysis buffer containing protease inhibitors. After incubation for 45 min
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at 4°C with rotation, 15 µl sheep anti-rabbit Dynabeads M-280 were added, and incubation
continued for 30 min at 4°C. The beads were washed with NP40 lysis buffer containing
protease inhibitors and 1% Triton X100. Bound proteins were eluted from the beads with
SDS-sample buffer, boiled for 5 min, and analysed by immunoblotting using anti-Flag, 1H9
or 4C8 antibodies.
Co-immunoprecipitations from mouse and human brain extracts were performed as described
(Sittler et al., 1998). Brain extracts were prepared and incubated with the anti-Atx-3 antibody
1H9 (1:500) and an anti-dynamin antibody (1:500). Antibody - protein complexes were
isolated and analysed by immunoblotting using anti-VCP and anti-Atx-3 (CT1) antibodies.
2. Figure Legends
Fig. S1
GST-pull down experiments with full length Atx-3 proteins containing a normal or mutated
VBM. GST-Atx-3 fusion proteins were bound to beads and incubated with His-VCP. After
washing, bound proteins were eluted and analysed by immunoblotting. The bottom panel
represents 10% of input material.
Fig. S2
BP43 at a concentration of 1 mM does not prevent the interaction between GST-VCP and
MBP-Ufd1.
Fig. S3
GST-pull down experiments with truncated Atx-3 proteins containing a normal or mutated
VBM. The experiments were carried out as described in S1.
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Fig. S4
Top, level of Atx-3 in one of the transgenic lines expressing the disease protein. Line #24.1
has ~17.25 ng Atx-3 protein/0.5 fly head (1 fly head is ~500 ng total protein). Bottom, level
of VCP in transgenic line #6.1, which has ~25 ng VCP/ 0.5 fly head, compared to the
standard. The ratio of VCP/Atx-3 is therefore ~1.45 for these flies. Data quantified with NIH
ImageJ.
3. References
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