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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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Pathogenic and substrate trap forms of torsinA display reduced mobility in the NE. (A) GFP immunolabeling of BHKGFPWT, BHKGFPΔE, and BHKGFPEQ stable cell lines. (B and D) GFP fluorescence of BHK21 cells transiently transfected with GFPWT-, GFPΔE-, or GFPEQ-torsinA and DsRed fluorescence of control cells transfected with DsRed2-ER (CLONTECH Laboratories, Inc.). Images show representative cells immediately before (top), immediately after (middle), and 120 s after (bottom) bleaching a ROI (boxed areas) in the NE (B) or ER (D). Bars, 10 μm. (C and E) Relative fluorescence intensity in the ROI as a function of time after photobleaching at time point “B” (B, bleach; see Materials and methods). Points show mean values and SEM.
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fig1: Pathogenic and substrate trap forms of torsinA display reduced mobility in the NE. (A) GFP immunolabeling of BHKGFPWT, BHKGFPΔE, and BHKGFPEQ stable cell lines. (B and D) GFP fluorescence of BHK21 cells transiently transfected with GFPWT-, GFPΔE-, or GFPEQ-torsinA and DsRed fluorescence of control cells transfected with DsRed2-ER (CLONTECH Laboratories, Inc.). Images show representative cells immediately before (top), immediately after (middle), and 120 s after (bottom) bleaching a ROI (boxed areas) in the NE (B) or ER (D). Bars, 10 μm. (C and E) Relative fluorescence intensity in the ROI as a function of time after photobleaching at time point “B” (B, bleach; see Materials and methods). Points show mean values and SEM.

Mentions: We have previously shown that, although wild-type (WT) torsinA is predominantly localized in the main ER, pathogenic ΔE-torsinA and a predicted “substrate trap” ATP hydrolysis-deficient EQ-torsinA concentrate in the NE (Fig. 1 A; Vale, 2000; Goodchild and Dauer, 2004). NE resident proteins typically concentrate in the nuclear membrane through a selective retention mechanism mediated by binding to the nuclear lamina (Burke and Stewart, 2002). Consequently, NE proteins are less mobile in the NE than in the ER membrane (Ellenberg et al., 1997). If torsinA interacts with a NE protein, it should therefore display similarly reduced mobility in the NE. We tested this concept by examining the mobility of torsinA using FRAP analysis of BHK21 cells transiently overexpressing GFPWT-, GFPΔE-, and GFPEQ-torsinA. At moderate expression levels, both GFPΔE- and GFPEQ-torsinA selectively localize in the NE (Fig. 1 B); these cells were used for NE FRAP measurements. Cells expressing higher levels of these proteins also contain fluorescence in the main ER (Fig. 1 D), allowing us to perform ER FRAP measurements. In the ER, all three forms of GFP-torsinA displayed a similar time course of fluorescence recovery (∼65% after 210 s; Fig. 1 E). In contrast, the NE fluorescence recovery of GFPΔE- and GFPEQ-torsinA was markedly slower than GFPWT-torsinA (Fig. 1 C). In the NE, only 50% of GFPΔE-torsinA and 40% of GFPEQ-torsinA fluorescence recovered within 330 s (Fig. 1 C), at which time 75% of GFPWT-torsinA fluorescence had returned. However, it is possible that contaminating fluorescence from ER GFPWT-torsinA may contribute to an overestimate of NE GFPWT-torsinA recovery.


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)

Pathogenic and substrate trap forms of torsinA display reduced mobility in the NE. (A) GFP immunolabeling of BHKGFPWT, BHKGFPΔE, and BHKGFPEQ stable cell lines. (B and D) GFP fluorescence of BHK21 cells transiently transfected with GFPWT-, GFPΔE-, or GFPEQ-torsinA and DsRed fluorescence of control cells transfected with DsRed2-ER (CLONTECH Laboratories, Inc.). Images show representative cells immediately before (top), immediately after (middle), and 120 s after (bottom) bleaching a ROI (boxed areas) in the NE (B) or ER (D). Bars, 10 μm. (C and E) Relative fluorescence intensity in the ROI as a function of time after photobleaching at time point “B” (B, bleach; see Materials and methods). Points show mean values and SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171781&req=5

fig1: Pathogenic and substrate trap forms of torsinA display reduced mobility in the NE. (A) GFP immunolabeling of BHKGFPWT, BHKGFPΔE, and BHKGFPEQ stable cell lines. (B and D) GFP fluorescence of BHK21 cells transiently transfected with GFPWT-, GFPΔE-, or GFPEQ-torsinA and DsRed fluorescence of control cells transfected with DsRed2-ER (CLONTECH Laboratories, Inc.). Images show representative cells immediately before (top), immediately after (middle), and 120 s after (bottom) bleaching a ROI (boxed areas) in the NE (B) or ER (D). Bars, 10 μm. (C and E) Relative fluorescence intensity in the ROI as a function of time after photobleaching at time point “B” (B, bleach; see Materials and methods). Points show mean values and SEM.
Mentions: We have previously shown that, although wild-type (WT) torsinA is predominantly localized in the main ER, pathogenic ΔE-torsinA and a predicted “substrate trap” ATP hydrolysis-deficient EQ-torsinA concentrate in the NE (Fig. 1 A; Vale, 2000; Goodchild and Dauer, 2004). NE resident proteins typically concentrate in the nuclear membrane through a selective retention mechanism mediated by binding to the nuclear lamina (Burke and Stewart, 2002). Consequently, NE proteins are less mobile in the NE than in the ER membrane (Ellenberg et al., 1997). If torsinA interacts with a NE protein, it should therefore display similarly reduced mobility in the NE. We tested this concept by examining the mobility of torsinA using FRAP analysis of BHK21 cells transiently overexpressing GFPWT-, GFPΔE-, and GFPEQ-torsinA. At moderate expression levels, both GFPΔE- and GFPEQ-torsinA selectively localize in the NE (Fig. 1 B); these cells were used for NE FRAP measurements. Cells expressing higher levels of these proteins also contain fluorescence in the main ER (Fig. 1 D), allowing us to perform ER FRAP measurements. In the ER, all three forms of GFP-torsinA displayed a similar time course of fluorescence recovery (∼65% after 210 s; Fig. 1 E). In contrast, the NE fluorescence recovery of GFPΔE- and GFPEQ-torsinA was markedly slower than GFPWT-torsinA (Fig. 1 C). In the NE, only 50% of GFPΔE-torsinA and 40% of GFPEQ-torsinA fluorescence recovered within 330 s (Fig. 1 C), at which time 75% of GFPWT-torsinA fluorescence had returned. However, it is possible that contaminating fluorescence from ER GFPWT-torsinA may contribute to an overestimate of NE GFPWT-torsinA recovery.

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