TorsinA in the nuclear envelope

Abstract

Early-onset torsion dystonia, a CNS-based movement disorder, is usually associated with a single amino acid deletion (Delta E302/303) in the protein torsinA. TorsinA is an AAA+ ATPase in the endoplasmic reticulum, but what it does is unknown. Here, we use torsinA mutants with defects in ATP hydrolysis (E171Q, ATP-bound) and ATP binding (K108A, ATP-free) to probe torsinA's normal cellular function. Surprisingly, ATP-bound torsinA is recruited to the nuclear envelope (NE) of transfected cells, where it alters connections between inner and outer nuclear membranes. In contrast, ATP-free torsinA is diffusely distributed throughout the endoplasmic reticulum and has no effect on the NE. Among AAA+ ATPases, affinity for substrates is high in the ATP-bound and low in the ATP-free state, leading us to propose that component(s) of the NE may be substrates for torsinA. We also find that the disease-promoting Delta E302/303 mutant is in the NE, and that this relocalization, as well as the mutant's previously described ability to induce membranous inclusions, is eliminated by the K108A ATP-binding mutation. These results suggest that changes in interactions involving torsinA in the NE could be important for the pathogenesis of dystonia and point to torsinA and related proteins as a class of ATPases that may operate in the NE.

Fig. 1.

Expression and distribution of torsinA mutants. (A) Domain structure of torsinA, showing position of Walker A (K108A), Walker B (E171Q), and DYT1 (ΔE302/303) mutations. N-terminal signal sequence (black), the adjacent hydrophobic domain implicated in ER retention and membrane association (residues 21–40, gray), and the position of our GFP tag are also shown. GFP was added to the C terminus to avoid interfering with the N-terminal hydrophobic sequences. (B) TorsinA-GFP mutants expressed in CHO cells are ER glycoproteins sensitive to endoglycosidase H digestion (compare - and + EndoH). (C) Localization of GFP-tagged torsinA mutants. Epifluorescence images show the distribution in transiently transfected COS-7 cells of wild type, K108A, E171Q torsinA-GFP, and torsinB(E178Q)-GFP. (D) Localization of untagged torsinA mutants. Shown are confocal images of CHO cells transiently transfected with untagged torsinA, torsinA(K108A), or torsinA(E171Q). TorsinA was visualized by indirect immunofluorescence. The first three images were taken with the same settings on the confocal microscope, whereas the fourth had reduced gain to show the distribution of highly overexpressed mutant. (E) Confocal images comparing the ER in CHO cells stably expressing torsinA(K108A)-GFP or (E171Q)-GFP with that in untransfected cells. Shown are GFP (Left), the ER marker protein disulfide isomerase (Center), and a merger of the two (Right).

Fig. 2.

Colocalization of torsinA mutants with NE markers. (A) Epifluorescence image of torsinA(K108A)-GFP and untransfected CHO cells stained for nucleoporin Nup153 (red). (B) Epifluorescence image of torsinA(E171Q)-GFP and untransfected CHO cells stained for Nup153 (red). (C) Confocal image of lower nuclear surface in (E171Q)-GFP expressing CHO cell stained for Nup153. Arrow highlights region lacking Nup153. (D) Confocal image of similar nuclear surface stained for LAP2.

Fig. 3.

NE ultrastructure in torsinA(E171Q)-GFP expressing CHO cells. (A) Control CHO NE (nucleus on top, cytoplasm below). (B) NE in torsinA(E171Q)-GFP cell line. Note regions of tight membrane apposition alternating with separated areas indicated by *. (C) Membranous inclusion in high-expressing torsinA(E171Q)-GFP cell line. (D) Control CHO NE. (E) Abnormal spacing in torsinA(E171Q)-GFP cell line. (F) Herniation of inner nuclear membrane into perinuclear space in torsinA(E171Q)-GFP cell line. (G) Quick-freeze/deep-etch image of CHO NE (nucleus on top, cytoplasm below). (H and I) Same in torsinA(E171Q)-GFP cell line. [Bars = 500 nm (A–E) and 100 nm (F).]

Fig. 4.

ΔE302/303-torsinA in the NE. (A) Indirect immunofluorescence showing localization of ΔE302/303-torsinA (untagged) in transfected CHO, PC12, and CAD cells. CHO low and CHO high show representative low and high expressing cells, taken with 4-fold differences in gain on the microscope. (B) Effects of K108A mutation on localization of ΔE302/303-torsinA. (Upper) COS-7 cells. (Lower) PC12 cells expressing wild-type torsinA (Left), ΔE302/303 torsinA (Center), and K108A/ΔE302/303 torsinA (Right). Cells were stained for torsinA (green) and PDI (red). (C) Inclusions formed by ΔE302/303-torsinA are enriched in lamin-B receptor-YFP. Shown are wild-type (Upper) and ΔE302/303 (Lower) torsinA-CFP coexpressed with lamin-B receptor-YFP. For merge, torsinA-CFP is green, and lamin-B receptor-YFP is red.