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. 2013 Apr 23;110(17):E1545-54.
doi: 10.1073/pnas.1300676110. Epub 2013 Apr 8.

Regulation of Torsin ATPases by LAP1 and LULL1

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Free PMC article

Regulation of Torsin ATPases by LAP1 and LULL1

Chenguang Zhao et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The LDs of LAP1 and LULL1 are necessary and sufficient for interaction with TorsinA in vivo. (A) An ATP arrested TorsinA mutant associates with LULL1. (Left) 293T cells were transfected with the indicated constructs, metabolically labeled with 35S methionine, and lysed in mild detergent 24 h posttransfection. Following immunoprecipitation with anti-HA antibodies, eluates were resolved by SDS/PAGE and visualized by autoradiography. CNX, calnexin. (Right) An immunoprecipitate obtained from TorAE171Q transfectants was eluted with SDS, diluted in buffer, and reimmunoprecipitated successively using anti-LULL1 and anti-TorA antibodies. The resulting immunoprecipitates were subjected to SDS/PAGE and autoradiography. Fifty percent of the eluate used for immunoprecipitation was loaded for comparison (input, left lane). (B) TorA E171Q associates with LAP1. (Left) 293T cells were transfected with the indicated constructs and subjected to an immunoprecipitation experiment as described in A. (Right) A reimmunoprecipitation experiment was performed as described above, using an anti-HA immunoprecipitate obtained from a TorA E171Q/FLAG-LAP1 double transfectant as starting material. Anti-TorA and anti-LAP1 antisera were used for reimmunoprecipitation. (C and D) Schematic view of LULL1 (C) and LAP1 (D) constructs used in this study. TM, transmembrane domain; SS, signal sequence. (E and F) The LDs of LULL1 and LAP1 associate with TorsinA. 293T cells were transfected with the indicated constructs, and subjected to mild detergent lysis and anti-HA immunoprecipitation. Input controls and immunoprecipitates were resolved by SDS/PAGE and subjected to immunoblotting using the indicated antibodies.
Fig. 2.
Fig. 2.
The physical association of LAP1 and LULL1 with TorsinA is direct and ATP-dependent. (A, Left) Purified LDs of LAP1 and LULL1 along with TorsinA or indicated mutant derivatives were subjected to SDS/PAGE and colloidal blue staining. (Right) Purified TorA WT was deglycosylated by incubation with PNGase F and subjected to SDS/PAGE and immunoblotting using anti-TorsinA antibodies. (B–E) Protein complex formation was monitored by size-exclusion chromatography. UV traces are shown in blue, elution positions of size markers are indicated by arrows on top. Elution fractions were subjected to immunoblotting using the indicated antibodies. (B) LDs of LAP1 or LULL1 were subjected to size-exclusion chromatography. (C) TorA E171Q was incubated with a twofold molar excess of LAP1LD or LULL1LD in the presence of 2 mM ATP and subjected to a Superdex 200 PC 3.2/30 column preequilibrated in 500 µM ATP. (D) TorA E171Q was incubated with a twofold molar excess of LAP1LD or LULL1LD in the absence of ATP during incubation or chromatographic separation. (E) TorA E171Q/ΔE was incubated with a twofold molar excess of LAP1LD or LULL1LD in presence of 2 mM ATP and analyzed as in B. Note that additional controls are shown in the supplement (Fig. S3).
Fig. 3.
Fig. 3.
LAP1 and LULL1 stimulate the ATPase activity of TorsinA. (A) TorsinA and its mutant derivatives were incubated in presence of 2 mM ATP, either alone or in presence of the LDs of LAP1LD or LULL1LD. Pi production as measure of ATP hydrolysis was monitored after 60 min using a malachite green assay. (B) Next, 3 µM TorA was incubated at with 3 µM LAP1LD or LULL1LD individually or a combination of both 1.5 µM LAP1LD and 1.5 µM LULL1LD. Pi release was monitored after 60 min. (C and D) Initial velocities of ATP hydrolysis were obtained after monitoring Pi release in presence of 3 µM TorsinA and increasing LAP1LD (C) or LULL1LD (D) concentrations as indicated. To increase the signal, the assay for LAP1LD was performed in 40 µL instead of 25 µL. The data were fitted to Michaelis–Menten kinetics in Prism and yielded the indicated apparent Km and Vmax values. (E and F) Job plot analysis to assess the stoichiometry of LAP1LD/LULL1LD binding to TorA. The total concentration of LAP1LD (or LULL1LD) and TorA in the ATPase assay was kept constant at 10 µM while varying the ratios of individual components.
Fig. 4.
Fig. 4.
Reconstitution of TorsinA in proteoliposomes. (A) The ATPase activity was measured for detergent-solubilized and reconstituted TorA. ER lipids (Methods) were added to detergent-solubilized TorA to a final concentration of 300 µM. (B) Proteoliposomes were incubated with increasing concentrations of proteinase K (PNK), in the absence or presence of Nonidet P-40, and subjected to SDS/PAGE and immunoblotting. (C) The amount of detergent-solubilized TorA and TorA in the proteoliposomes used in above ATPase assay was compared via SDS/PAGE and colloidal blue staining.
Fig. 5.
Fig. 5.
LAP1 and LULL1 are not unfolded by TorsinA. Native LAP1LD or LULL1LD were incubated in presence of GroEL D87K and an ATP-regenerating system, or in the additional presence of TorA. After 30 min, the reaction mixtures were applied onto a Superdex 200 PC column and eluted with trap buffer. Fractions were collected and separated by SDS/PAGE, and subjected to immunoblotting using antisera against LAP1 or LULL1. The UV elution profile of GroEL D87K and the positions of size markers are included for reference. As positive control, LAP1LD or LULL1LD were denatured in 8 M urea, and rapidly diluted 1:50 into otherwise identical reactions.
Fig. 6.
Fig. 6.
LAP1LD and LULL1LD specifically accelerate the ATP hydrolysis step of the ATPase cycle. (A and B) Single turnover kinetics of TorA/B ATPases alone (basal rate) or in presence of the indicated cofactor. Data represent a mean obtained from three independent experiments. Error bars indicate the SD. (C) Rate constants obtained from fitting the data above to a single exponential decay function, using Prism. (D) Stimulation of TorA/B by LAP1LD/LULL1LD relative to the basal rate as observed in A and B.
Fig. 7.
Fig. 7.
Regulation of the Torsin family by LAP1 and LULL1. Three micromolars of each Torsin was incubated in presence of 2 mM ATP, either alone or in presence of the LDs of LAP1 or LULL1. Pi production as measure of ATP hydrolysis was monitored after 60 min using a malachite green assay.

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