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. 2007 Apr 24;104(17):7271-6.
doi: 10.1073/pnas.0701185104. Epub 2007 Apr 11.

Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells

Jeffrey W Hewett  1 , Bakhos Tannous, Brian P Niland, Flavia C Nery, Juan Zeng, Yuqing Li, Xandra O Breakefield
Affiliations

Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells

Jeffrey W Hewett et al. Proc Natl Acad Sci U S A. .

Abstract

TorsinA is an AAA(+) protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope responsible for early onset torsion dystonia (DYT1). Most cases of this dominantly inherited movement disorder are caused by deletion of a glutamic acid in the carboxyl terminal region of torsinA. We used a sensitive reporter, Gaussia luciferase (Gluc) to evaluate the role of torsinA in processing proteins through the ER. In primary fibroblasts from controls and DYT1 patients most Gluc activity (95%) was released into the media and processed through the secretory pathway, as confirmed by inhibition with brefeldinA and nocodazole. Fusion of Gluc to a fluorescent protein revealed coalignment and fractionation with ER proteins and association of Gluc with torsinA. Notably, fibroblasts from DYT1 patients were found to secrete markedly less Gluc activity as compared with control fibroblasts. This decrease in processing of Gluc in DYT1 cells appear to arise, at least in part, from a loss of torsinA activity, because mouse embryonic fibroblasts lacking torsinA also had reduced secretion as compared with control cells. These studies demonstrate the exquisite sensitivity of this reporter system for quantitation of processing through the secretory pathway and support a role for torsinA as an ER chaperone protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Validation of Gluc secretion assay, linearity with cell number and time. Control (C2, circles) and DYT1 (D3, squares) fibroblasts were infected with a lentivirus vector encoding Gluc-IRES-cerulean to achieve infection of >90% cells. (A) Infected cells were plated 48 h after infection at different cell numbers per well, and luciferase activity in the medium was quantitated 24 h after plating. (B) Infected cells were plated at a density of 2.5 × 103 cells per well, and luciferase activity in the media was quantitated 24, 48, and 72 h after plating. Mean values are shown ± SD; comparison of control and DYT1 secretion at 72 h; ∗, P < 0.001.
Fig. 2.
Fig. 2.
Rate of Gluc secretion in DYT1 fibroblasts as compared with control fibroblasts. Cells from three DYT1 and three control lines were infected with the lentivirus vector encoding Gluc-IRES-cerulean and replated 48 h after infection at 2.5 × 103 cells per well. (A) Gluc activity 24 h after plating was determined in the medium as RLU per cell per 24 h. The experiment was repeated two times in triplicate for each cell line, shown as mean ± SD. The average mean of DYT1 lines and control lines was significantly different at P < 0.004. (B and C) Examples of the high infectivity of control (C1) and DYT1 (D3) lines as assessed by fluorescence. (Magnification ×100.) (Scale bars, 10 μM.)
Fig. 3.
Fig. 3.
Levels and distribution of Gluc-YFP in control and DYT1 media and cells. Cells were infected with lentivirus vector encoding Gluc-YFP, and, 24 h later, cells were plated at 2.5 × 103 per well. (A and B) Twenty-four hours after plating, luciferase activity was assessed in the media (A) or in living cells (B). In both control (C1) and DYT1 (D2) cells ≈5% activity was retained in cells with 95% in the media. (C) Western blot analysis indicated similar levels of Gluc-YFP protein in both cell types by using α-tubulin as an index of loading. Note that this was repeated in two additional DYT1 (D1, D4) and one control line (C2) with similar results.
Fig. 4.
Fig. 4.
Differential solubilization of control and DYT1 fibroblasts expressing Gluc-YFP. Twenty-four hours after infection with Gluc-YFP-IRES-cerulean lentivirus vector monolayers of control (C1) (Left) and DYT1 (D1) (Right) cells were homogenized directly (lysates) or sequentially extracted with digitonin (cytoplasmic fraction), Triton X-100 (ER fraction), and SDS/scraping (remaining proteins). Lysate and extracts were resolved by SDS/PAGE and immunoblotted for the cytoplasmic marker GAPDH, ER markers calnexin and torsinA, and the reporter protein Gluc-YFP. Note that this was repeated in two additional DYT1 (D2, D4) and one control lines (C2) with similar results.
Fig. 5.
Fig. 5.
ER distribution of Gluc-YFP and PDI in control and DYT1 cells. Control (C1) and DYT1 (D1) cells were infected with Gluc-YFP lentivirus vector, and, 24 h later, cells were treated on ice with digitonin. Dual immunocytochemistry was carried out for Gluc-YFP (B and F) and the ER marker PDI (C and G) with merged images shown in the rightmost panels (D and H). Nuclei were stained with DAPI (A and E). (Magnification ×100.) (Scale bars, 10 μm.)
Fig. 6.
Fig. 6.
Coimmune precipitation of torsinA and Gluc-YFP. Control (C4) (Left) and DYT1 (D2) (Right) cells were infected with lentivirus vector encoding Gluc-YFP-IRES-cerulean or YFP-IRES-cerulean (control), and, 72 h later, lysates were prepared and immune precipitated (IP) for torsinA. Lysates and torsinA-IP pellets were resolved by SDS/PAGE, and Western blotting was carried out with antibodies to GFP.
Fig. 7.
Fig. 7.
Gluc secretion in torsinA knockout MEFs. MEFs in early passages were genotyped by genomic PCR as described (39). Cells were infected with lentivirus vector encoding Gluc-IRES-cerulean and replated 48 h later at 2.5 × 103 cells per well, and Gluc in media was assessed 24 h later. The experiment was repeated two times in triplicate for each genotype and the mean is shown ±SD. The mean values for torsinA+/+ versus torsinA−/− cells were significantly different: ∗, P < 0.001.
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