Figure 3. Absence of torsinA or overexpression of variant forms of torsinA impairs ER exit of YFP-VSVG.

293 T cells transfected with torsinA siRNA (si1958 and si1963) or scrambled siRNA as a control were infected with adenovirus expressing YFP-tagged VSVG_ts045 (VSVG) and incubated at 40.5°C for 16 hr. Following this incubation, cells were either fixed immediately (time 0 - ER loaded VSVG), or transferred to a 32°C water bath and trafficking of VSVG was assessed. (A) In scrambled control cells, ER loaded VSVG (0 min) can be visualized (green fluorescence) as it traffics in vesicles (10 min, arrow heads) to the Golgi complex (20 min) where the protein temporarily accumulates while undergoing N-glycosylation (20 min, arrow heads). Trafficking of VSVG from the ER to the Golgi complex showed a defect in ER exit following loss of torsinA (arrows). (B) Knock-down efficiency was confirmed after 72 hr by western analysis. Quantification of ER associated VSVG shows a decrease in rate of VSVG exit from the ER following torsinA silencing after 10 min (C) and 20 min (D). (E) Quantification of Golgi complex-associated VSVG shows a decrease in rate of Golgi complex accumulation following torsinA silencing (n=3). In addition, uninfected 293T cells, and 293T cells expressing torsinAwt, and variant forms: torsinAΔE, torsinAR288Q and torsinAF205I were infected with adenovirus expressing YFP-tagged VSVG_ts045 (VSVG) and incubated, as described above. (F) Representative fluorescent images of cells stained for torsinA (red) and YFP-VSVG (green). (G) Image analysis quantification of the ER localization of VSVG in cells expressing different torsinA isoforms after 10 min; and (H) after 20 min at 32°C. Expression of variant torsinA forms are associated with delayed exit of VSVG from the ER in 293T cells after 10 and 20 min. Quantification of Golgi complex-associated VSVG shows a decrease in the rate of Golgi complex accumulation in torsinA variants (n=4). (Scale bars = 10 µm, ANOVA post-hoc Dunnett's t-test *p<0.05, **p<0.01).