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The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein.

Goodchild RE, Dauer WT - J. Cell Biol. (2005)

Bottom Line: Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the DeltaE mutation enhances this interaction.Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA.Interestingly, LULL1 resides in the main ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Columbia University, New York, NY 10032, USA.

ABSTRACT
A glutamic acid deletion (DeltaE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the DeltaE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of "substrate trap" EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.

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TorsinA interacts with LULL1. (A) TorsinA interacts with the conserved lumenal domain of LULL1. Immunofluorescent labeling of transfected BHKGFPEQ cells with anti-GFP and anti-myc antibodies. Full-length LULL1 (top) and the LULL1 lumenal domain (bottom) recruit GFPEQ-torsinA to the ER. (B) TorsinA coimmunoprecipitates with myc-LULL1. Immunoprecipitations and immunoblotting were performed as in Fig. 2 F except that transfections were performed with myc-LULL1. Immunoglobulin heavy chains were not visible with the exposure time needed to visualize GFPEQ-torsinA. (C) RT-PCR of mouse LAP1 and LULL1 from whole tissue RNA. (D) Rabbit polyclonal antibodies against LAP1, LULL1, and torsinA similarly detect their respective antigens. BHK21 cells were transfected with myc-tagged mouse forms of LAP1, LULL1, and torsinA; and WCL was probed with anti-myc to confirm that similar amounts of transfected protein were loaded (top panel). Immunoblots were subsequently probed (bottom panel) with anti-LAP1, anti-LULL1, and anti-torsinA at concentrations that generated similar levels of immunoreactivity. Comparative images are from a simultaneous exposure of a single immunoblot. (E) Immunoblots of NIH-3T3 WCL probed with rabbit polyclonal antibodies. 15 μg of 1% SDS NIH-3T3 WCL were probed with rabbit polyclonal antibodies at the concentrations used in D. Images are from a simultaneous 2-s exposure of a single immunoblot. (F) NIH-3T3 cells transfected with GFPWT-torsinA (left) or GFPEQ-torsinA (right) and labeled with anti-GFP. (G) LAP1 and LULL1 interact more strongly with substrate trap EQ-torsinA. WCL were prepared from BHKGFPWT cells (Cell line: WT) and BHKGFPEQ cells (Cell line: EQ) transfected with myc-LAP1 (Tfct: LAP1) or myc-LULL1 (Tfct: LULL1). Proteins were immunoprecipitated from WCL with anti-myc antibody, eluted from protein G agarose beads and immunoblotted. Parallel control precipitations were performed in the absence of anti-myc antibody. Immunoblots of immunoprecipitated proteins and 2% of WCL were probed with anti-torsinA and anti-myc. WCL from BHKGFPWT cells contained more GFP-torsinA, myc-LAP1, and myc-LULL1 than BHKGFPEQ cells because this was necessary to visualize coprecipitated GFPWT-torsinA. The position of the 65-kD GFP-torsinA is indicated by an arrow (top). Neither myc-LAP1, myc-LULL1, or GFP-torsinA proteins were immunoprecipitated in the absence of anti-myc antibody.
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fig5: TorsinA interacts with LULL1. (A) TorsinA interacts with the conserved lumenal domain of LULL1. Immunofluorescent labeling of transfected BHKGFPEQ cells with anti-GFP and anti-myc antibodies. Full-length LULL1 (top) and the LULL1 lumenal domain (bottom) recruit GFPEQ-torsinA to the ER. (B) TorsinA coimmunoprecipitates with myc-LULL1. Immunoprecipitations and immunoblotting were performed as in Fig. 2 F except that transfections were performed with myc-LULL1. Immunoglobulin heavy chains were not visible with the exposure time needed to visualize GFPEQ-torsinA. (C) RT-PCR of mouse LAP1 and LULL1 from whole tissue RNA. (D) Rabbit polyclonal antibodies against LAP1, LULL1, and torsinA similarly detect their respective antigens. BHK21 cells were transfected with myc-tagged mouse forms of LAP1, LULL1, and torsinA; and WCL was probed with anti-myc to confirm that similar amounts of transfected protein were loaded (top panel). Immunoblots were subsequently probed (bottom panel) with anti-LAP1, anti-LULL1, and anti-torsinA at concentrations that generated similar levels of immunoreactivity. Comparative images are from a simultaneous exposure of a single immunoblot. (E) Immunoblots of NIH-3T3 WCL probed with rabbit polyclonal antibodies. 15 μg of 1% SDS NIH-3T3 WCL were probed with rabbit polyclonal antibodies at the concentrations used in D. Images are from a simultaneous 2-s exposure of a single immunoblot. (F) NIH-3T3 cells transfected with GFPWT-torsinA (left) or GFPEQ-torsinA (right) and labeled with anti-GFP. (G) LAP1 and LULL1 interact more strongly with substrate trap EQ-torsinA. WCL were prepared from BHKGFPWT cells (Cell line: WT) and BHKGFPEQ cells (Cell line: EQ) transfected with myc-LAP1 (Tfct: LAP1) or myc-LULL1 (Tfct: LULL1). Proteins were immunoprecipitated from WCL with anti-myc antibody, eluted from protein G agarose beads and immunoblotted. Parallel control precipitations were performed in the absence of anti-myc antibody. Immunoblots of immunoprecipitated proteins and 2% of WCL were probed with anti-torsinA and anti-myc. WCL from BHKGFPWT cells contained more GFP-torsinA, myc-LAP1, and myc-LULL1 than BHKGFPEQ cells because this was necessary to visualize coprecipitated GFPWT-torsinA. The position of the 65-kD GFP-torsinA is indicated by an arrow (top). Neither myc-LAP1, myc-LULL1, or GFP-torsinA proteins were immunoprecipitated in the absence of anti-myc antibody.

Mentions: We transfected myc-LULL1 into BHKGFPEQ cells to determine if this ER-localized LAP1 homologue also interacts with torsinA. Consistent with this notion, myc-LULL1 produced a clear redistribution of GFPEQ-torsinA from the NE to the ER and there was strong colocalization between GFP and myc labeling in transfected cells (Fig. 5 A). We obtained similar results with a LULL1 fragment containing only the transmembrane and lumenal domains (208LULL1; Fig. 5 A), confirming that this domain is responsible for the effects observed with full-length LULL1. In addition, GFPEQ-torsinA coimmunoprecipitates with myc-LULL1 from lysates of myc-LULL1–transfected BHKGFPEQ cells (Fig. 5 B). Together, these results suggest that LULL1 interacts with torsinA in the main ER. Like torsinA, LAP1 and LULL1 mRNAs are widely expressed in both neural and nonneural tissue (Fig. 5 C), which is consistent with the hypothesis that these proteins may be physiologically relevant interactors of torsinA.


The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein.

Goodchild RE, Dauer WT - J. Cell Biol. (2005)

TorsinA interacts with LULL1. (A) TorsinA interacts with the conserved lumenal domain of LULL1. Immunofluorescent labeling of transfected BHKGFPEQ cells with anti-GFP and anti-myc antibodies. Full-length LULL1 (top) and the LULL1 lumenal domain (bottom) recruit GFPEQ-torsinA to the ER. (B) TorsinA coimmunoprecipitates with myc-LULL1. Immunoprecipitations and immunoblotting were performed as in Fig. 2 F except that transfections were performed with myc-LULL1. Immunoglobulin heavy chains were not visible with the exposure time needed to visualize GFPEQ-torsinA. (C) RT-PCR of mouse LAP1 and LULL1 from whole tissue RNA. (D) Rabbit polyclonal antibodies against LAP1, LULL1, and torsinA similarly detect their respective antigens. BHK21 cells were transfected with myc-tagged mouse forms of LAP1, LULL1, and torsinA; and WCL was probed with anti-myc to confirm that similar amounts of transfected protein were loaded (top panel). Immunoblots were subsequently probed (bottom panel) with anti-LAP1, anti-LULL1, and anti-torsinA at concentrations that generated similar levels of immunoreactivity. Comparative images are from a simultaneous exposure of a single immunoblot. (E) Immunoblots of NIH-3T3 WCL probed with rabbit polyclonal antibodies. 15 μg of 1% SDS NIH-3T3 WCL were probed with rabbit polyclonal antibodies at the concentrations used in D. Images are from a simultaneous 2-s exposure of a single immunoblot. (F) NIH-3T3 cells transfected with GFPWT-torsinA (left) or GFPEQ-torsinA (right) and labeled with anti-GFP. (G) LAP1 and LULL1 interact more strongly with substrate trap EQ-torsinA. WCL were prepared from BHKGFPWT cells (Cell line: WT) and BHKGFPEQ cells (Cell line: EQ) transfected with myc-LAP1 (Tfct: LAP1) or myc-LULL1 (Tfct: LULL1). Proteins were immunoprecipitated from WCL with anti-myc antibody, eluted from protein G agarose beads and immunoblotted. Parallel control precipitations were performed in the absence of anti-myc antibody. Immunoblots of immunoprecipitated proteins and 2% of WCL were probed with anti-torsinA and anti-myc. WCL from BHKGFPWT cells contained more GFP-torsinA, myc-LAP1, and myc-LULL1 than BHKGFPEQ cells because this was necessary to visualize coprecipitated GFPWT-torsinA. The position of the 65-kD GFP-torsinA is indicated by an arrow (top). Neither myc-LAP1, myc-LULL1, or GFP-torsinA proteins were immunoprecipitated in the absence of anti-myc antibody.
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fig5: TorsinA interacts with LULL1. (A) TorsinA interacts with the conserved lumenal domain of LULL1. Immunofluorescent labeling of transfected BHKGFPEQ cells with anti-GFP and anti-myc antibodies. Full-length LULL1 (top) and the LULL1 lumenal domain (bottom) recruit GFPEQ-torsinA to the ER. (B) TorsinA coimmunoprecipitates with myc-LULL1. Immunoprecipitations and immunoblotting were performed as in Fig. 2 F except that transfections were performed with myc-LULL1. Immunoglobulin heavy chains were not visible with the exposure time needed to visualize GFPEQ-torsinA. (C) RT-PCR of mouse LAP1 and LULL1 from whole tissue RNA. (D) Rabbit polyclonal antibodies against LAP1, LULL1, and torsinA similarly detect their respective antigens. BHK21 cells were transfected with myc-tagged mouse forms of LAP1, LULL1, and torsinA; and WCL was probed with anti-myc to confirm that similar amounts of transfected protein were loaded (top panel). Immunoblots were subsequently probed (bottom panel) with anti-LAP1, anti-LULL1, and anti-torsinA at concentrations that generated similar levels of immunoreactivity. Comparative images are from a simultaneous exposure of a single immunoblot. (E) Immunoblots of NIH-3T3 WCL probed with rabbit polyclonal antibodies. 15 μg of 1% SDS NIH-3T3 WCL were probed with rabbit polyclonal antibodies at the concentrations used in D. Images are from a simultaneous 2-s exposure of a single immunoblot. (F) NIH-3T3 cells transfected with GFPWT-torsinA (left) or GFPEQ-torsinA (right) and labeled with anti-GFP. (G) LAP1 and LULL1 interact more strongly with substrate trap EQ-torsinA. WCL were prepared from BHKGFPWT cells (Cell line: WT) and BHKGFPEQ cells (Cell line: EQ) transfected with myc-LAP1 (Tfct: LAP1) or myc-LULL1 (Tfct: LULL1). Proteins were immunoprecipitated from WCL with anti-myc antibody, eluted from protein G agarose beads and immunoblotted. Parallel control precipitations were performed in the absence of anti-myc antibody. Immunoblots of immunoprecipitated proteins and 2% of WCL were probed with anti-torsinA and anti-myc. WCL from BHKGFPWT cells contained more GFP-torsinA, myc-LAP1, and myc-LULL1 than BHKGFPEQ cells because this was necessary to visualize coprecipitated GFPWT-torsinA. The position of the 65-kD GFP-torsinA is indicated by an arrow (top). Neither myc-LAP1, myc-LULL1, or GFP-torsinA proteins were immunoprecipitated in the absence of anti-myc antibody.
Mentions: We transfected myc-LULL1 into BHKGFPEQ cells to determine if this ER-localized LAP1 homologue also interacts with torsinA. Consistent with this notion, myc-LULL1 produced a clear redistribution of GFPEQ-torsinA from the NE to the ER and there was strong colocalization between GFP and myc labeling in transfected cells (Fig. 5 A). We obtained similar results with a LULL1 fragment containing only the transmembrane and lumenal domains (208LULL1; Fig. 5 A), confirming that this domain is responsible for the effects observed with full-length LULL1. In addition, GFPEQ-torsinA coimmunoprecipitates with myc-LULL1 from lysates of myc-LULL1–transfected BHKGFPEQ cells (Fig. 5 B). Together, these results suggest that LULL1 interacts with torsinA in the main ER. Like torsinA, LAP1 and LULL1 mRNAs are widely expressed in both neural and nonneural tissue (Fig. 5 C), which is consistent with the hypothesis that these proteins may be physiologically relevant interactors of torsinA.

Bottom Line: Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the DeltaE mutation enhances this interaction.Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA.Interestingly, LULL1 resides in the main ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Columbia University, New York, NY 10032, USA.

ABSTRACT
A glutamic acid deletion (DeltaE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the DeltaE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of "substrate trap" EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.

Show MeSH
Related in: MedlinePlus