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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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LAP1 recruits torsinA to the NE. (A) GFPWT-torsinA in BHKGFPWT cells transfected with different NE proteins. Arrows show transfected GFPWT-torsinA cells expressing candidate NE proteins, which were identified by colabeling for myc or β-galactosidase reporters (see online supplemental material for more details). (B) Immunolabeling of myc-LAP1–transfected BHKGFPWT cells with anti-myc and anti-GFP. (C) Immunolabeling of myc-LAP1–transfected BHK21 cells with anti-myc and anti-PDI. (D) GFP fluorescence of HeLa cells transiently transfected with GFPEQ-torsinA and GFP-LAP1 immediately before (prebleach), immediately after (postbleach), and at 180 and 360 s after photobleaching in the ROI (boxed area). (E) Relative fluorescence intensity in the ROI as a function of time after photobleaching. FRAP analysis was performed as described in Materials and methods. Points represent mean and SEM. (F) Coimmunoprecipitation of GFPEQ-torsinA with myc-LAP1. Immunoprecipitations with anti-myc antibody were performed with whole cell lysates (WCL) of BHKGFPEQ cells (Cell line: EQ) or BHK21 cells (Cell line: BHK) transfected with myc-LAP1. Immunoblots of WCL and immunoprecipitated proteins were probed with anti-torsinA (top panel) and anti-myc (bottom panel). GFPEQ-torsinA coimmunoprecipitates with myc-LAP1 from transfected BHKGFPEQ cells (position of GFPEQ-torsinA indicated by arrow) but not in the absence of anti-myc antibody (second lane), with mock-transfected BHKGFPEQ cells (third lane), or with myc-LAP1–transfected BHK21 cells (fourth lane). The position of immunoglobulin heavy chains is indicated (Ig, arrowhead).
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fig2: LAP1 recruits torsinA to the NE. (A) GFPWT-torsinA in BHKGFPWT cells transfected with different NE proteins. Arrows show transfected GFPWT-torsinA cells expressing candidate NE proteins, which were identified by colabeling for myc or β-galactosidase reporters (see online supplemental material for more details). (B) Immunolabeling of myc-LAP1–transfected BHKGFPWT cells with anti-myc and anti-GFP. (C) Immunolabeling of myc-LAP1–transfected BHK21 cells with anti-myc and anti-PDI. (D) GFP fluorescence of HeLa cells transiently transfected with GFPEQ-torsinA and GFP-LAP1 immediately before (prebleach), immediately after (postbleach), and at 180 and 360 s after photobleaching in the ROI (boxed area). (E) Relative fluorescence intensity in the ROI as a function of time after photobleaching. FRAP analysis was performed as described in Materials and methods. Points represent mean and SEM. (F) Coimmunoprecipitation of GFPEQ-torsinA with myc-LAP1. Immunoprecipitations with anti-myc antibody were performed with whole cell lysates (WCL) of BHKGFPEQ cells (Cell line: EQ) or BHK21 cells (Cell line: BHK) transfected with myc-LAP1. Immunoblots of WCL and immunoprecipitated proteins were probed with anti-torsinA (top panel) and anti-myc (bottom panel). GFPEQ-torsinA coimmunoprecipitates with myc-LAP1 from transfected BHKGFPEQ cells (position of GFPEQ-torsinA indicated by arrow) but not in the absence of anti-myc antibody (second lane), with mock-transfected BHKGFPEQ cells (third lane), or with myc-LAP1–transfected BHK21 cells (fourth lane). The position of immunoglobulin heavy chains is indicated (Ig, arrowhead).

Mentions: Based on the behavior of WT and mutant torsinA, we next sought to identify a torsinA NE binding partner. We developed a screening procedure based on the assumption that overexpressing a NE-localized torsinA substrate would increase the amount of torsinA in the NE, which is normally quite low. We selected candidate proteins that normally reside in the NE and contain a predicted lumenal domain that is conserved between mammalian species because these features indicate a potential functional role within the NE lumen. Cells stably expressing GFPWT-torsinA (BHKGFPWT; Fig. 1 A) were transfected with 18 candidate protein cDNAs in a reporter plasmid that coexpresses β-galactosidase (Table I and Fig. 2 A). Of all tested NE candidate proteins, only LAP1 recruited GFPWT-torsinA to the NE in a uniform perinuclear distribution reminiscent of substrate trap GFPEQ-torsinA (Table I and Fig. 2 A; compare transfected and untransfected cells). Occasionally, cells expressing high levels of lamin B receptor, LUMA, and Sun2 contained bright puncta of GFPWT-torsinA. These puncta were considered to be a nonspecific effect of gross overexpression because they were randomly located in the NE and ER. We further examined the LAP1 recruitment of GFPWT-torsinA by expressing myc-tagged LAP1 (myc-LAP1) in BHKGFPWT cells. As expected, cells expressing myc-LAP1 concentrated GFPWT-torsinA in the NE (Fig. 2 B), whereas the unrelated ER chaperone, protein disulphide isomerase (PDI), was unaltered (Fig. 2 C).


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)

LAP1 recruits torsinA to the NE. (A) GFPWT-torsinA in BHKGFPWT cells transfected with different NE proteins. Arrows show transfected GFPWT-torsinA cells expressing candidate NE proteins, which were identified by colabeling for myc or β-galactosidase reporters (see online supplemental material for more details). (B) Immunolabeling of myc-LAP1–transfected BHKGFPWT cells with anti-myc and anti-GFP. (C) Immunolabeling of myc-LAP1–transfected BHK21 cells with anti-myc and anti-PDI. (D) GFP fluorescence of HeLa cells transiently transfected with GFPEQ-torsinA and GFP-LAP1 immediately before (prebleach), immediately after (postbleach), and at 180 and 360 s after photobleaching in the ROI (boxed area). (E) Relative fluorescence intensity in the ROI as a function of time after photobleaching. FRAP analysis was performed as described in Materials and methods. Points represent mean and SEM. (F) Coimmunoprecipitation of GFPEQ-torsinA with myc-LAP1. Immunoprecipitations with anti-myc antibody were performed with whole cell lysates (WCL) of BHKGFPEQ cells (Cell line: EQ) or BHK21 cells (Cell line: BHK) transfected with myc-LAP1. Immunoblots of WCL and immunoprecipitated proteins were probed with anti-torsinA (top panel) and anti-myc (bottom panel). GFPEQ-torsinA coimmunoprecipitates with myc-LAP1 from transfected BHKGFPEQ cells (position of GFPEQ-torsinA indicated by arrow) but not in the absence of anti-myc antibody (second lane), with mock-transfected BHKGFPEQ cells (third lane), or with myc-LAP1–transfected BHK21 cells (fourth lane). The position of immunoglobulin heavy chains is indicated (Ig, arrowhead).
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fig2: LAP1 recruits torsinA to the NE. (A) GFPWT-torsinA in BHKGFPWT cells transfected with different NE proteins. Arrows show transfected GFPWT-torsinA cells expressing candidate NE proteins, which were identified by colabeling for myc or β-galactosidase reporters (see online supplemental material for more details). (B) Immunolabeling of myc-LAP1–transfected BHKGFPWT cells with anti-myc and anti-GFP. (C) Immunolabeling of myc-LAP1–transfected BHK21 cells with anti-myc and anti-PDI. (D) GFP fluorescence of HeLa cells transiently transfected with GFPEQ-torsinA and GFP-LAP1 immediately before (prebleach), immediately after (postbleach), and at 180 and 360 s after photobleaching in the ROI (boxed area). (E) Relative fluorescence intensity in the ROI as a function of time after photobleaching. FRAP analysis was performed as described in Materials and methods. Points represent mean and SEM. (F) Coimmunoprecipitation of GFPEQ-torsinA with myc-LAP1. Immunoprecipitations with anti-myc antibody were performed with whole cell lysates (WCL) of BHKGFPEQ cells (Cell line: EQ) or BHK21 cells (Cell line: BHK) transfected with myc-LAP1. Immunoblots of WCL and immunoprecipitated proteins were probed with anti-torsinA (top panel) and anti-myc (bottom panel). GFPEQ-torsinA coimmunoprecipitates with myc-LAP1 from transfected BHKGFPEQ cells (position of GFPEQ-torsinA indicated by arrow) but not in the absence of anti-myc antibody (second lane), with mock-transfected BHKGFPEQ cells (third lane), or with myc-LAP1–transfected BHK21 cells (fourth lane). The position of immunoglobulin heavy chains is indicated (Ig, arrowhead).
Mentions: Based on the behavior of WT and mutant torsinA, we next sought to identify a torsinA NE binding partner. We developed a screening procedure based on the assumption that overexpressing a NE-localized torsinA substrate would increase the amount of torsinA in the NE, which is normally quite low. We selected candidate proteins that normally reside in the NE and contain a predicted lumenal domain that is conserved between mammalian species because these features indicate a potential functional role within the NE lumen. Cells stably expressing GFPWT-torsinA (BHKGFPWT; Fig. 1 A) were transfected with 18 candidate protein cDNAs in a reporter plasmid that coexpresses β-galactosidase (Table I and Fig. 2 A). Of all tested NE candidate proteins, only LAP1 recruited GFPWT-torsinA to the NE in a uniform perinuclear distribution reminiscent of substrate trap GFPEQ-torsinA (Table I and Fig. 2 A; compare transfected and untransfected cells). Occasionally, cells expressing high levels of lamin B receptor, LUMA, and Sun2 contained bright puncta of GFPWT-torsinA. These puncta were considered to be a nonspecific effect of gross overexpression because they were randomly located in the NE and ER. We further examined the LAP1 recruitment of GFPWT-torsinA by expressing myc-tagged LAP1 (myc-LAP1) in BHKGFPWT cells. As expected, cells expressing myc-LAP1 concentrated GFPWT-torsinA in the NE (Fig. 2 B), whereas the unrelated ER chaperone, protein disulphide isomerase (PDI), was unaltered (Fig. 2 C).

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