Arresting a Torsin ATPase reshapes the endoplasmic reticulum

Abstract

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.

Keywords: AAA+ ATPase; ATPases; Electron Microscopy (EM); Endoplasmic Reticulum(ER); Enzyme Mechanisms; Membrane Structure; Torsin.

FIGURE 1.

Overexpression of a TorsinB dominant-negative “trap” mutant induces cytoplasmic foci. A, ATPase activity of TorsinB wild type in the absence or presence of activating cofactor LULL1 and the dominant-negative TorsinB E178Q mutant. Phosphate release was measured using a malachite green assay after 1 h as described previously (9) B, TorsinB E178Q dominant-negative mutant expressed from HeLa-derived stable cell lines by addition of 500 ng/ml doxycycline. Immunoblot was probed with anti-TorsinB antibody. Control cells are parental cells of the protein-producing stable cell line containing only the “Tet-On” plasmid. Bands corresponding to TorsinB and its glycosylated forms are indicated. Arrowhead: TorsinB degradation product. Numbers on the left refer to molecular mass in kilodaltons. C, immunofluorescent images of same stable cell line as in B uninduced or induced with 500 ng/ml doxycycline. D, single cell analysis of foci formation in TorsinB E178Q overexpressing cells (black bars) compared with TorsinB wild type-overexpressing cells (gray bars) after 24 h treatment with doxycycline to overexpress their respective proteins. (*): p < 0.05. E, immunofluorescent images of TorsinB E178Q cell line induced with 500 ng/ml doxycycline for 24 h and costained with antibodies against endogenous LULL1 or LAP1. All cells were stained with anti-TorsinB antibody. Scale bar: 10 μm.

FIGURE 2.

Characterization of TorsinB E178Q foci with compartmental markers. A, immunofluorescent images of transiently transfected TorsinB E178Q-HA (TorBEQ-HA) or TorsinB E178Q overexpressed from doxycycline-induced stable cell lines (TorBEQ) stained with standard ER markers calnexin, PDI, Sec61, and KDEL. B, immunofluorescent images of transiently transfected TorsinB E178Q-HA (TorBEQ-HA) stained with antibodies against inner nuclear membrane proteins LaminB1, Sun2, and Emerin. Green: TorsinB or HA; Red: compartmental marker proteins; Scale bar: 10 μm.

FIGURE 3.

Reorganization of the endoplasmic reticulum by a TorsinB trap variant. A, EM image of control parental “Tet-On” cells showing normal ER and nuclear envelope morphology. B, low magnification image of HeLa-derived stable cell line overexpressing TorsinB E178Q showing a reorganization of cytoplasmic membranes into sinusoidal ER structures. C, high magnification image of section depicted in B showing a perinuclear vesicle (arrowhead). D, EM image of a representative isolated cytosolic focus from TorsinB E178Q overexpressing cells induced with 500 ng/ml doxycycline for 24 h. E, EM image showing connectivity of TorB E178Q-induced structures. Sinusoidal ER membranes can be seen forming as branches emanating from ER tubules (arrows). F, schematic representation of sinusoidal ER structures. The arrow marks an analogous position to the arrow in panel E. N: nucleus. NPC, nuclear pore complex.

FIGURE 4.

TorsinB E178Q immunostaining. A, low magnification image of HeLa-derived stable cell line overexpressing TorsinB E178Q immunostained with anti-TorsinB antibody and proteinA conjugated to gold. B, higher magnification image of section depicted in A. C, high magnification image of membrane structures associated with the overexpression of TorsinB E178Q. Gold particle diameter is 10 nm.

FIGURE 5.

Effect of overexpressing TorsinB E178Q on MHC-I processing. A, autoradiogram of MHC-I immunoprecipitated from TorsinB E178Q or control cell lines at 0, 30, or 60 min post radiolabeling with ³⁵S. Each sample was treated to remove high mannose N-glycans (EndoH) or all glycans (PNGaseF) as acquisition of EndoH resistance is a measure of transport of MHC-I through the golgi. B, supernatants from A were immunoprecipitated with anti-TorsinB antibody to confirm overexpression. Numbers on the left refer to molecular mass in kilodaltons.

FIGURE 6.

Dependence of TorsinB E178Q foci on the presence of LULL1. A, representative images of cells from TorsinB E178Q stable cell lines either uninduced or induced with 500 ng/ml doxycycline for 24 h. siLULL1 is treated with siRNA against LULL1 for 48 h pre-induction with doxycycline. B, quantitation of number of foci-positive cells in the conditions used in A. Each bar represents the mean of three separate samples of 100–110 cells each. Error bars: ± one S.D. C, immunoblot of samples from conditions as in A and B showing knockdown of LULL1 and overexpression TorsinB E178Q. (*): nonspecific bands

FIGURE 7.

Effect of C-terminal aromatic residue of TorsinB on LULL1 binding. A, domain diagram of TorsinB. Sequence comparison showing very C-terminal amino acids with boxed aromatic amino acid conserved between all human Torsins. Line demarcates beginning of truncation for TorBΔ334 construct. B, immunoprecipitation of LULL1^LD-HA with various TorsinB constructs including the C-terminal mutants. Numbers on the left refer to molecular mass in kilodaltons. C, ATPase activity of TorsinB WT (circles) versus TorsinB-GGG (triangles) in the presence of varying concentrations of LULL1. Phosphate release was measured using a malachite green assay as described previously (9). D, ATPase activity of TorsinB WT (circles) versus TorsinB-GGG (triangles) in the presence of varying concentrations of LAP1. Phosphate release was measured using a malachite green assay as described previously (9). Kinetic parameters are adjacent to their corresponding curves.

FIGURE 8.

Effect of TorsinB C-terminal deletion on foci formation. A, immunofluorescent images of TorB E178Q Δ334 in the absence or presence of 500 ng/ml doxycycline for 24 h. B, single cell analysis of foci formation in TorB E178Q cell line (black bars) versus TorB E178Q Δ334 cell line (gray bars) after 24 h treatment with 500 ng/ml doxycycline to induce overexpression of their respective proteins. (*): p < 0.05.