Open Access

Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA

  • Hui Li1,
  • Hui-Chuan Wu1,
  • Zhonghua Liu1, 3,
  • Lucia F Zacchi2,
  • Jeffrey L Brodsky2 and
  • Michal Zolkiewski1Email author

Received: 21 October 2014

Accepted: 9 December 2014

Published: 16 December 2014


A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.


Early-onset dystonia, TorsinA AAA+ ATPase Protein association


Early-onset torsion dystonia (EOTD) is the most common and severe form of primary dystonia, a neurological disorder that manifests as uncontrollable movements and abnormal body postures. Most cases of EOTD are associated with a deletion of a single GAG codon in the DYT1 gene. As a result, a single glutamic acid residue is absent in the EE pair located in the C-terminal region of torsinA (Ozelius et al. 1997). TorsinA is a putative member of the AAA+ superfamily of A TPases a ssociated with different a ctivities (Neuwald et al. 1999). The torsinA mRNA is widely expressed in various human tissues, including the central nervous system, but the biological role of torsinA is not completely clear (reviewed in (Tanabe et al. 2009; Zolkiewski and Wu 2011)). AAA+ ATPases are energy-driven “molecular machines”, which remodel the conformation of macromolecules and disassemble macromolecular complexes (Hanson and Whiteheart 2005). Proteins from the AAA+ family form ring-shaped hexameric complexes, which enclose their substrate molecules. Hexamer formation is essential for the activity of AAA+ ATPases (Barnett et al. 2000). Numerous torsinA partners have been identified and the association with some of these is compromised when the mutant gene product is expressed (Naismith et al. 2009; Zolkiewski and Wu 2011). However, the identity of a torsinA substrate that is critical for its cellular activity is unknown and, more fundamentally, whether torsinA even forms a hexamer in cells has not been fully established. Moreover, it is unclear how the glutamate deletion affects these biochemical properties and in turn, which defect(s) associated with the mutant protein are linked to EOTD.

The torsinA sequence contains an N-terminal ER-targeting signal peptide that is cleaved upon import into the ER lumen, producing the mature 36-kDa form of the protein (Liu et al. 2003). The signal sequence is followed by a 20-residue-long hydrophobic segment that is responsible for membrane association (Liu et al. 2003) and ER retention (Vander Heyden et al. 2011). The AAA+ module of torsinA is located downstream of the membrane-binding domain and contains a non-canonical ATP-binding Walker-A motif (Nagy et al. 2009; Zolkiewski and Wu 2011) and six cysteines that are absent from other AAA+ ATPases (Zhu et al. 2008; Zolkiewski and Wu 2011). The site of the dystonia-linked glutamate deletion (E302/E303) is located within the C-terminal AAA+ subdomain, which supports oligomerization of other AAA+ ATPases (Barnett et al. 2000). Studies with purified recombinant torsinA revealed either a monomeric protein (Kustedjo et al. 2003; Zhu et al. 2008) or a spectrum of high-molecular weight particles (Zhao et al. 2013). In contrast, torsinA assemblies ranging from monomers and dimers to hexamers were detected in lysates from mammalian cells (Kustedjo et al. 2000; Gordon and Gonzalez-Alegre 2008; Vander Heyden et al. 2009; Jungwirth et al. 2010). How these assemblies are affected by the disease-causing mutation or the hydrophobic membrane anchor has not yet been established.

To this end, we investigated the size of human torsinA complexes after isolation from cultured mammalian cells. We found that the main oligomeric species is consistent with the formation of torsinA hexamers, but this structure becomes less stable when the dystonia-linked protein variant is expressed. We also found that the membrane-bound hydrophobic segment stabilizes the torsinA oligomer. These data add fundamental new insights to our understanding of torsinA structure and suggest why the loss of a single amino acid can exhibit profound cellular effects.

Results and discussion

To determine whether human torsinA and the dystonia-linked torsinAΔE variant oligomerize in the cell, we expressed each protein in two cell lines, HEK293 and CHO cells. After preparation of cell lysates in dodecylmaltoside, BN-PAGE and immunoblotting with an anti-torsinA antibody was used to observe the distribution of the torsinA-containing species (Figure 1). Both stably transfected cell lines produced comparable amounts of torsinA and torsinAΔE (Figure 1A, B, lower panels). In addition to some monomeric torsinA and torsinAΔE (shown by the bands below 66 kDa), BN-PAGE detected a single major immunoreactive species migrating close to the 200-kDa complex of β-amylase, but slower than the 242-kDa protein standard (Figure 1A, B, upper panels). The migration of the torsinA oligomer in BN-PAGE is consistent with that of a homohexamer (predicted molecular weight 216 kDa) and is consistent with the formation of a species of similar size in BN-PAGE using lysates prepared from U2OS cells (Vander Heyden et al. 2009). It cannot be excluded, however, that the detected species corresponds to a hetero-oligomer containing torsinA and other components, such as the torsinA binding partners LAP1 and LULL1 (Goodchild and Dauer 2005; Zhao et al. 2013; Sosa et al. 2014). We also found that the deletion of Glu302 in torsinA apparently destabilizes the oligomeric species (Figure 1A, B, upper panels). This result suggests that the dystonia-linked torsinA variant may be defective in either self-association or interactions with other proteins. Indeed, the efficiency of torsinAΔE interaction with LAP1 and LULL1 is compromised relative to the wild type protein (Naismith et al. 2009; Zhao et al. 2013). In contrast, the dystonia-linked torsinAΔE variant shows an enhanced binding affinity for nesprin (Nery et al. 2008). Thus, the apparent loss of the detected oligomeric species in the torsinAΔE producing cells (Figure 1) suggests that the observed torsinA complex does not include nesprin. Nevertheless, the data presented in Figure 1 indicate that the EOTD-associated mutation has a profound effect on oligomer and/or complex formation, and we propose that this defect might impact the protein’s function and disease presentation.
Figure 1

BN-PAGE analysis of torsinA complexes. Full-length human torsinA (WT) or the dystonia-linked torsinAΔE protein (ΔE) was expressed in HEK293 (A) and CHO (B) cells. Production of the torsinA variants was confirmed by SDS-PAGE followed by immunoblotting with anti-torsinA antibodies (lower panels) using untransfected cells as a control (C). The cell lysates were separated on BN-PAGE followed by immunoblotting (upper panels). For BN-PAGE, the migration positions of the native-electrophoresis standards are indicated. The migration position of β-amylase (200 kDa) is indicated with an arrow. Protein migration in BN-PAGE can reflect other biophysical properties, besides the molecular weight, so the molecular weight determination is only approximate. The figure shows a representative result from two independent experiments.

As noted above, previous studies on purified full-length torsinA failed to detect oligomeric species, which could have been the result of detergent-induced destabilization of intersubunit contacts (Kustedjo et al. 2003). Other experiments using a purified protein detected higher molecular weight species that were not further resolved (Zhao et al. 2013). To obtain enriched soluble protein in the absence of a detergent, we produced a truncated torsinA variant lacking the hydrophobic membrane-binding region, torsinAΔ40 (Liu et al. 2003). TorsinAΔ40 was expressed in S2 cells and purified from the culture media (see Materials and Methods). Interestingly, torsinAΔ40ΔE was poorly secreted in S2 culture (Liu et al. 2003), which is consistent with the apparent mislocalization of this dystonia variant from the ER lumen to the nuclear envelope (Goodchild and Dauer 2004; Naismith et al. 2004). The circular dichroism spectrum of torsinAΔ40 (Figure 2A) was similar to that of the full-length torsinA purified with detergent (Kustedjo et al. 2003), which indicates that a deletion of the hydrophobic segment does not inhibit folding of torsinA. TorsinAΔ40 was strictly monomeric (~30 kDa, Figure 2B), regardless of whether size exclusion chromatography was run in the absence of nucleotides or in the presence of ATP or ADP, which in other cases stabilize AAA+ hexamers (Akoev et al. 2004).
Figure 2

Oligomerization of the N-terminally truncated torsinA variants. (A) Far-UV circular dichroism spectra of purified torsinAΔ40 (1 mg/ml, solid line) and the dialysis buffer (dotted line) are shown. (B) Gel-filtration analysis of torsinAΔ40 in the absence of nucleotides or in the presence of 2 mM ATP or ADP is shown. The elution times of molecular weight standards (kDa) are indicated. (C) BN-PAGE (upper panel) and SDS-PAGE (lower panel) analysis was followed by immunoblotting with anti-torsinA antibodies of lysates from HEK293 and CHO cells expressing either full-length torsinA (WT), torsinAΔ40 (Δ40), torsinAΔ40ΔE (Δ40ΔE) or untransfected cells (C). For BN-PAGE, the migration positions of the native-electrophoresis standards are indicated. The migration position of β-amylase (200 kDa) is indicated with an arrow. Protein migration in BN-PAGE can reflect other biophysical properties, besides the molecular weight, so the molecular weight determination is only approximate. The figure shows a representative result from two independent experiments.

To corroborate these data, we next investigated the oligomeric state of torsinAΔ40 in mammalian cell lysates (Figure 2C). In contrast to the full-length protein (WT), the torsinAΔ40 and torsinAΔ40ΔE variants again failed to form oligomeric species in BN-PAGE. This result is in accordance with the properties of purified torsinAΔ40 (Figure 2B) and indicates that the 20 residue-long N-terminal hydrophobic segment is essential to stabilize torsinA complexes. Two mechanisms can be proposed to account for this result. First, the hydrophobic segment may directly participate in either self-association or hetero-association with another protein’s membrane-embedded domain. Second, the membrane association of torsinA and its retention in the ER lumen may increase the likelihood of forming the homo- or heterooligomers. Recently, hetero-hexamers of torsinA and LAP1 were reconstituted with purified proteins (Sosa et al. 2014). Since LAP1 is a transmembrane protein, its interaction with torsinA in the cell might be efficient only if torsinA is also targeted to the ER membrane by its N-terminal hydrophobic segment. Future efforts will be directed to address this hypothesis.


In summary, we found that torsinA forms a discrete high-molecular weight complex in mammalian cells. However, the complex is destabilized by the dystonia-linked mutation, but is stabilized by the membrane anchor. Establishing a link between defects in torsinAΔE oligomerization and EOTD will be an important focus of future research efforts.


Plasmids, antibodies, and reagents

DNA constructs containing the human torsinA sequence in pcDNA3 vector were described before (Liu et al. 2003) and anti-torsin antibodies were obtained as described (Zacchi et al. 2014). Sweet potato β-amylase was from Sigma. Native electrophoresis protein standards were from Invitrogen/Life Technologies.

Cell culture

HEK293 cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (BioWhittaker) at 37°C in the presence of 5% CO2. CHO-K1 cells (ATCC) were maintained in F-12 K medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells were transfected with pcDNA3 expression vectors containing the torsinA variants using FuGene 6 transfection reagent (Roche) according to the manufacturer’s instructions. Stably transfected cells were selected in the presence of 1 mg/mL G418 (Invitrogen).

Blue-native PAGE

BN-PAGE is a native gel electrophoresis technique, where the Coomassie Brilliant Blue dye binds to membrane protein complexes and provides the electric charge for the electrophoretic separation. BN-PAGE was carried out as previously described (Wittig et al. 2006; Vander Heyden et al. 2009; Jungwirth et al. 2010). Briefly, ~90% confluent cells were collected and solubilized in a lysis buffer (50 mM imidazole pH 7.0, 50 mM NaCl, 2 mM 6-aminohexanoic acid, 4 mM MgCl2, 2 mM EDTA, 2 mM ATP, 1 mM PMSF, and 0.25% dodecylmaltoside) for 15 min in 4°C, and centrifuged twice for 15 min at 15,000 rpm in IEC Micromax benchtop centrifuge. The supernatant supplemented with 0.0625% Coomassie blue G-250 and 5% glycerol was loaded onto a 9% polyacrylamide gel. Following electrophoresis, separated proteins were transferred onto PVDF membrane and subjected to immunoblotting using anti-torsinA antibodies, followed by horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Southern Biotechnology). Signal detection was performed with WestPico chemiluminescence kit (Pierce).

Protein purification

The S2 cell line stably transfected with a torsinAΔ40 expression plasmid containing the BiP signal sequence followed by an N-terminal His-tag was produced as previously described (Liu et al. 2003). Cells were grown at 23°C in S2 medium (Invitrogen) supplemented with 0.5% DMSO when the density reached 107 cells/ml and protein expression was induced after 24 h with 0.7 mM CuSO4. The cells were separated from the culture medium by centrifugation 6 days post induction. The medium was filtered through a 0.45 μm membrane and loaded onto a 2.5-ml Chelating Sepharose column (GE Healthcare). Proteins were eluted with an imidazole step concentration gradient. Fractions containing torsinAΔ40 (eluted at 10–50 mM imidazole) were pooled, concentrated on Centriplus YM-10 (Millipore), and dialyzed in 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 20 mM MgCl2, and 10% glycerol.

Circular dichroism spectroscopy

CD spectra were measured with a Jasco J-720 spectrometer using a 0.01-cm cylindrical cuvette at room temperature.

Gel filtration chromatography

Gel filtration analysis was performed at room temperature with a Shimadzu HPLC. TorsinAΔ40 samples (20 μl, ~0.2 mg/ml) were analyzed at 0.04 ml/min on a Superdex 200 PC 3.2/30 column (GE Healthcare) equilibrated in 50 mM Tris/HCl pH 7.5, 0.2 M KCl, 20 mM MgCl2 without nucleotides or with 2 mM ATP or ADP.



This research was supported by a grant (to JLB and MZ) and a postdoctoral fellowship (to LFZ) from the Dystonia Medical Research Foundation and by grant GM75061 from the National Institutes of Health to JLB. This is contribution 15-105-J from the Kansas Agricultural Experiment Station. Publication of this article was funded in part by the Kansas State University Open Access Publishing Fund.

Authors’ Affiliations

Department of Biochemistry and Molecular Biophysics, Kansas State University
Department of Biological Sciences, University of Pittsburgh
Department of Embryology, Carnegie Institution


  1. Akoev V, Gogol EP, Barnett ME, Zolkiewski M: Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB. Protein Sci 2004, 13: 567-574. 10.1110/ps.03422604View ArticleGoogle Scholar
  2. Barnett ME, Zolkiewska A, Zolkiewski M: Structure and activity of ClpB from escherichia coli. Role of the amino-and -carboxyl-terminal domains. J Biol Chem 2000, 275: 37565-37571. 10.1074/jbc.M005211200View ArticleGoogle Scholar
  3. Goodchild RE, Dauer WT: Mislocalization to the nuclear envelope: an effect of the dystonia-causing torsinA mutation. Proc Natl Acad Sci U S A 2004, 101: 847-852. 10.1073/pnas.0304375101View ArticleGoogle Scholar
  4. Goodchild RE, Dauer WT: The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein. J Cell Biol 2005, 168: 855-862. 10.1083/jcb.200411026View ArticleGoogle Scholar
  5. Gordon KL, Gonzalez-Alegre P: Consequences of the DYT1 mutation on torsinA oligomerization and degradation. Neuroscience 2008, 157: 588-595. 10.1016/j.neuroscience.2008.09.028View ArticleGoogle Scholar
  6. Hanson PI, Whiteheart SW: AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol 2005, 6: 519-529. 10.1038/nrm1684View ArticleGoogle Scholar
  7. Jungwirth M, Dear ML, Brown P, Holbrook K, Goodchild R: Relative tissue expression of homologous torsinB correlates with the neuronal specific importance of DYT1 dystonia-associated torsinA. Hum Mol Genet 2010, 19: 888-900. 10.1093/hmg/ddp557View ArticleGoogle Scholar
  8. Kustedjo K, Bracey MH, Cravatt BF: Torsin A and its torsion dystonia-associated mutant forms are lumenal glycoproteins that exhibit distinct subcellular localizations. J Biol Chem 2000, 275: 27933-27939.Google Scholar
  9. Kustedjo K, Deechongkit S, Kelly JW, Cravatt BF: Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant delta E-torsinA. Biochemistry 2003, 42: 15333-15341. 10.1021/bi0349569View ArticleGoogle Scholar
  10. Liu Z, Zolkiewska A, Zolkiewski M: Characterization of human torsinA and its dystonia-associated mutant form. Biochem J 2003, 374: 117-122. 10.1042/BJ20030258View ArticleGoogle Scholar
  11. Nagy M, Wu HC, Liu Z, Kedzierska-Mieszkowska S, Zolkiewski M: Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB. Protein Sci 2009, 18: 287-293. 10.1002/pro.36View ArticleGoogle Scholar
  12. Naismith TV, Heuser JE, Breakefield XO, Hanson PI: TorsinA in the nuclear envelope. Proc Natl Acad Sci U S A 2004, 101: 7612-7617. 10.1073/pnas.0308760101View ArticleGoogle Scholar
  13. Naismith TV, Dalal S, Hanson PI: Interaction of torsinA with its major binding partners is impaired by the dystonia-associated DeltaGAG deletion. J Biol Chem 2009, 284: 27866-27874. 10.1074/jbc.M109.020164View ArticleGoogle Scholar
  14. Nery FC, Zeng J, Niland BP, Hewett J, Farley J, Irimia D, Li Y, Wiche G, Sonnenberg A, Breakefield XO: TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton. J Cell Sci 2008, 121: 3476-3486. 10.1242/jcs.029454View ArticleGoogle Scholar
  15. Neuwald AF, Aravind L, Spouge JL, Koonin EV: AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 1999, 9: 27-43.Google Scholar
  16. Ozelius LJ, Hewett JW, Page CE, Bressman SB, Kramer PL, Shalish C, de Leon D, Brin MF, Raymond D, Corey DP, Fahn S, Risch NJ, Buckler AJ, Gusella JF, Breakefield XO: The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein. Nat Genet 1997, 17: 40-48. 10.1038/ng0997-40View ArticleGoogle Scholar
  17. Sosa BA, Demircioglu FE, Chen JZ, Ingram J, Ploegh H, Schwartz TU: How lamina-associated polypeptide 1 (LAP1) activates torsin. eLife 2014, 3: e03239.View ArticleGoogle Scholar
  18. Tanabe LM, Kim CE, Alagem N, Dauer WT: Primary dystonia: molecules and mechanisms. Nat Rev Neurol 2009, 5: 598-609. 10.1038/nrneurol.2009.160View ArticleGoogle Scholar
  19. Vander Heyden AB, Naismith TV, Snapp EL, Hodzic D, Hanson PI: LULL1 retargets TorsinA to the nuclear envelope revealing an activity that is impaired by the DYT1 dystonia mutation. Mol Biol Cell 2009, 20: 2661-2672. 10.1091/mbc.E09-01-0094View ArticleGoogle Scholar
  20. Vander Heyden AB, Naismith TV, Snapp EL, Hanson PI: Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum. EMBO J 2011, 30: 3217-3231. 10.1038/emboj.2011.233View ArticleGoogle Scholar
  21. Wittig I, Braun HP, Schagger H: Blue native PAGE. Nat Protoc 2006, 1: 418-428. 10.1038/nprot.2006.62View ArticleGoogle Scholar
  22. Zacchi LF, Wu HC, Bell SL, Millen L, Paton AW, Paton JC, Thomas PJ, Zolkiewski M, Brodsky JL: The BiP molecular chaperone plays multiple roles during the biogenesis of torsinA, an AAA+ ATPase associated with the neurological disease early-onset torsion dystonia. J Biol Chem 2014, 289: 12727-12747. 10.1074/jbc.M113.529123View ArticleGoogle Scholar
  23. Zhao C, Brown RS, Chase AR, Eisele MR, Schlieker C: Regulation of Torsin ATPases by LAP1 and LULL1. Proc Natl Acad Sci U S A 2013, 110: E1545-E1554. 10.1073/pnas.1300676110View ArticleGoogle Scholar
  24. Zhu L, Wrabl JO, Hayashi AP, Rose LS, Thomas PJ: The torsin-family AAA+ protein OOC-5 contains a critical disulfide adjacent to Sensor-II that couples redox state to nucleotide binding. Mol Biol Cell 2008, 19: 3599-3612. 10.1091/mbc.E08-01-0015View ArticleGoogle Scholar
  25. Zolkiewski M, Wu HC: Emerging area: TorsinA, a novel ATP-dependent factor linked to dystonia, in protein chaperones and protection from neurodegenerative diseases. Edited by: Witt SN. Hoboken, New Jersey: John Wiley & Sons; 2011.Google Scholar


© Li et al.; licensee Springer. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.