COS-7 cells were transfected with fusion proteins and analyzed by fluorescence microscopy and immunocytochemistry 48 h later. (a–c): The following fusion proteins were expressed individually and appeared in a reticular pattern: (a) mCherry-Derlin-1 (b) His-p97 and (c) Myc-VIMP. (d-f): Cells were co-transfected with expression cassettes for (d) mCherry-Derlin-1 and (e) torsinA and 48 h later were processed for immunocytochemistry and fluorescence microscopy. These two overexpressed proteins co-localized in all cells in a reticular pattern, as seen in (f) merged image. (g-n): cells were co-transfected with expression cassettes for mCherry-Derlin-1, His-p97, torsinA and Myc-VIMP. Forty-eight h later proteins were visualized as follows: (g & k) mCherry-Derlin-1, (h) His-p97, (i & m) torsinA, and (l) Myc-VIMP, with (j & n) merged images showing colocalization in puncta. Representative cells are shown. Magnification bar = 10 μm.

Neuronal cultures were prepared from E15 mouse embryos. (a-f) Established cultures were infected overnight with (a-c) AAV-CBA-torsinA-GFP or (d-f) AAV-CBA-torsinAΔE-GFP at an M.O.I. of 50 genome copies per cell. Seventy h later immunocytochemistry was performed for (a & d) torsinA and (b & e) Derlin-1, with merged images shown in panels (c) and (f), respectively. Representative neurons shown. Magnification bar = 10 μm. (g) Neurons expressing only endogenous proteins were harvested and lysates immunoprecipitated with IgG or antibodies to mouse torsinA. Immune precipitates were resolved by SDS-PAGE and immunoblotted with antibodies to p97, mouse torsinA and Derlin-1. In the total lysate (input) so little immunoreactive Derlin-1 observed after the 2 min exposure that the blot had to be re-exposed for 90 min to visualize this protein (see inset box in input lane). For Derlin-1 the top band has the correct MW for this protein, an additional immunoreactive lower band was seen only in torsinA immune precipitates from neuronal cultures.

(a) Immunoblot showing >95% suppression of torsinA expression in COS cells treated with siRNA 1958 or 1963 for torsinA, with scr. siRNA as control. (b & c): (b) After siRNA transfection cells were intoxicated with wild-type CT for 45 min at 37°C and then fractionated into cytosolic and membrane components which were resolved by SDS-PAGE. Immunoblotting was carried out with antibodies to CTA1, CTB, BiP, β-actin and Hsp90. (c) Bar graph showing relative band densities of CTA1 in the cytosol normalized to Hsp90 in the cytosol, and CTA1 in the membrane fraction normalized to BiP. Results are shown as the mean of 3 experiments ± S.D.; * represents p <0.05 compared to the negative control siRNA transfected cells by Student’s t-test. (d) Immunoblot of post-ConA eluates and total input for the pull-down from control or torsinA siRNA cells intoxicated with CTB-GS for 3 h at 37°C. Arrowheads indicate the glycosylated, higher-MW form. Non-glycosylated CTB-GS is also present. (e) A fraction of the post-ConA eluates from (d) were analyzed by SDS-PAGE and silver staining. (f) Intoxicated cells were analyzed for the percentage of cells demonstrating fluorescently labeled CTB at the NE [boxed area]. Magnification bar = 5 μm. (g) Cells labeled with CTB at the NE were counted by three independent investigators in a blinded manner and calculated as mean ± SEM, and shown as percentage of positive staining of NE from the total cell number counted; 161 cells were counted for the control siRNA and 177 cells were counted for torsinA siRNA. No statistically significant difference (p>0.05) was noted between scr. control and torsinA siRNA as evaluated by the Student’s t-test.

(a & b) Primary skin fibroblasts from two DYT1 patients (dashed lines; D2551 =Δ; D2306 = ∇) and two controls (solid lines; HF19 = circle; 2131 = square) were infected with a lentivirus vector carrying the expression cassette for Gluc followed 72 h later by exposure to varying concentrations of (a) DTT or (b) 6-OHDA for 4 h. The activity of Gluc in the medium was measured using a luminometer^15,34. DTT treatment results are shown as the mean of 3 experiments ± S.D. at 0.625 mM concentration (where control lines gave essentially equal values). The difference between D2551 and controls was *p<0.011 and between D2306 and controls, ** p<0.003 using Student t-test. 6-OHDA results are shown as the mean of 3 experiments ± S.D. at 10 μM concentration where both DYT1 and control lines each gave essentially equal values (* p<0.005 comparing DYT1 and control lines using Student’s t-test). (c) Representative western blot showing control fibroblast line, HF77 and DYT1 fibroblast line, D2551, each co-infected with adenovirus vectors expressing GFP (to control for infection efficiency) and GFP-CFTRΔF508. (d) Band densities of GFP-CFTRΔF508 in three experiments using control fibroblast lines HF19 and HF77, and DYT1 fibroblast lines D2306 and D2551 were normalized to GFP (level of infection) and β-actin (loading control) and expressed as the mean ± S.D. The average of the control lines was compared with the average of the DYT1 lines (* p<0.005 using Student’s t-test).

(a–i) COS-7 cells were co-transfected with His-p97, Myc-VIMP and torsinA and visualized by immunocytochemistry 48 hrs later. His-p97 and Myc-VIMP formed puncta (a & d, respectively), while torsinA remained distributed in a reticular pattern (b & e); as shown in (c & f) merged images. (g-i) These same co-transfected cells were also stained for (g) His-p97 in puncta and (h) PDI in a reticular pattern, with (i) showing merged images. Representative cells shown. Magnification bar = 10 μm. (j) The microsomal fraction was isolated from 293T cells, solubilized (input) and immunoprecipitated at 4°C with antibodies to p97 or IgG (rabbit alpha). The precipitate was resolved by SDS-PAGE and immunoblotted to antibodies to p97, torsinA, Derlin-1 and VIMP. (* 25 kDa band in VIMP blot designates IgG.) (k) 293T cells were transfected with an 80 μM of a scr. siRNA or an siRNA targeted to the Derlin-1 mRNA for 72 h. Cell lysates were treated with digitonin to remove cytoplasmic proteins and the remaining membrane fractions were resuspended in RIPA buffer and immune precipitated with IgG or antibodies to p97. The solubilized membrane fraction (input) and immune precipitates were analyzed by SDS-PAGE and immunoblotted using antibodies to p97, torsinA or Derlin-1.

(a) 293T cells transfected with an expression cassette for GFP-CFTRΔF508 and 48 h later cell lysates were immunoprecipitated with IgG or antibodies to CFTR or torsinA, followed by SDS-PAGE and immunoblotting for GFP and torsinA. (b & c): (b) 293T cells were co-transfected with expression cassettes for GFP-CFTRΔF508 or torsinA or torsinAΔE or torsinAΔC or with the control cassette, pcDNA 3.1. Twenty-four h later samples were treated or not with MG132 for 16 h. Seventy-two h post-transfection, cell lysates were processed by SDS-PAGE and gels immunoblotted with antibodies to GFP, torsinA and β-actin. Representative immunoblots are shown with short (1 min) and long (3 min) exposures for GFP, and with 3 min exposure for torsinA and β-actin. (c) GFP-CFTRΔF508 band densities were normalized to those of torsinA and β-actin and represented as the mean of 3 experiments ± S.D. For statistical comparisons, * denotes the comparisons of control to torsinA or torsinAΔE or torsinAΔC transfections (p,<0.001, p<0.006, p<0.020, respectively) and + the comparisons of torsinA to torsinAΔE and torsinAΔC transfected cells (p<0.038, p<0.010, respectively) using Student’s t-test. (d & e): (d) 293T cells were transfected with 50 nM scr. siRNA or siRNAs for torsinA (1958 or 1963). Twenty-four h later cells were transfected with GFP-CFTRΔF508 in the absence or presence of MG132. Forty-eight h later cell lysates were resolved by SDS-PAGE and immunoblotted for GFP, torsinA and β-actin. (e) GFP-CFTRΔF508 band densities were normalized to those of torsinA and β-actin. Results are presented as the mean of 3 experiments ± S.D. The p values (determined by the Student’s t-test) were * p<0.009 and ** p<0.001 for comparison of scr. siRNA to siRNAs 1958 and 1963, respectively. (f & g): (f) 293T cells were transfected with 100 nM scr. siRNA or siRNA for Derlin-1. Twenty-four h later cells were transfected with a cassette for GFP-CFTRΔF508 alone or together with torsinA or GFP. Forty-eight h after the second transfection cell lysates were resolved by SDS-PAGE and immunoblotted for GFP, torsinA, Derlin-1 and β-actin. (g) Band densities of GFP-CFTRΔF508 normalized to torsinA and β-actin bands from 3 separate experiments are presented as the mean ± S.D. (* p=0.0006, ** p=0.05, *** p=0.048) using Student’s t-test.

(a) Fluorescent micrographs of tunicamycin treated C. elegans strains carrying the ER stress reporter hsp-4::GFP alone with a transgene of intestinally expressed mutant CFTRΔF508, or mutant CFTRΔF508 and wild-type human torsinA. The images depict the anterior region of C. elegans, while the boxed areas indicating the region measured within all animals. Magnification bar = 100 μm (b) Bar charts of GFP intensity from the stress reporter hsp-4::GFP alone, with wild-type or mutant CFTR ΔF508, and/or with wild-type torsinA. GFP pixel intensity measurements were consistently performed in the region boxed in (a). Data presented has been normalized to untreated hsp-4::GFP samples as the mean ± S.D. of three experiments, where 30 animals were analyzed per replicate (*p<0.05, Student’s t-test). (c) Transgene expression was evaluated by semi-quantitative real-time PCR (RT-PCR) using primers to amplify ama-1 (control) and CFTR mRNAs. Normalized intensity values for the various transgenic lines are displayed below the blot images. Multi Gauge (Version 3.0) used for statistical analysis showed no difference (p>0.05).

The cartoon represents the ER-localized misfolded integral membrane protein CFTRΔF508 whose post-translational assembly has been arrested due to a mutation in the NBD1 domain. The global alterations in CFTRΔF508 structure caused by the deletion of residue F508 are denoted by differences in the shape of the NBD domains, with the NBD1 mutated domain depicted by a starburst shape. This misfolded protein is detected by cytosolic chaperone/recognition factors and ubiquitinylated by ubiquitin ligases (E1, E2 and E3; for simplicity, not all known factors of each component class are depicted). The ubiquitinylation of CFTRΔF508 is required in this pathway for the extraction and delivery of CFTRΔF508 out of the ER membrane to the cytosolic proteasome. It is likely that torsinA cooperates with E3 ubiquitin ligases, such as RMA1and gp78 associated with the Derlin complex, and not with the Hrd-1 complex⁵⁸ to facilitate export of CFTRΔF508 from the ER. The putative Derlin-1 channel is labeled with a question mark, as it is not clear if this is the translocon used by this mutant protein. Other components of the translocon complex represented in the cartoon are VIMP and p97. Activated ubiquitin is transferred from E1 to E2 ubiquitin ligases. Following transfer, E2 covalently binds the activated ubiquitin to the target substrate which has been recognized by E3 ligase forming a polyubiquitin chain. Polyubiquitylated CFTRΔF508 protein is attached and recognized by both the N-domain (N) of p97 and the cofactor Ufd1/Npl4 (U/N). (Figure modified from Ye et al.²⁷; Turnbull et al.⁵⁹; Hebert et al.⁶⁰).