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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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LULL1 is an ER resident protein with homology to LAP1. (A) The percentage of amino acid sequence identity of predicted nucleoplasmic (LAP1), cytoplasmic (LULL1), and transmembrane and lumenal portions of human LULL1 and LAP1. (B) CLUSTAL W alignment of human LAP1 and LULL1 amino acid sequences. Asterisk indicates position with identical amino acid residues, colon indicates conserved amino acid residues, and period indicates semi-conserved amino acid residues. Predicted membrane spanning domains (determined with TMPred) are shaded and a conserved potential N-linked glycosylation site is boxed. (C) BHK21 cells transfected with myc-LULL1 were lysed in buffer with or without 1% Triton X-100 and centrifuged to separate lysates into soluble (S) and insoluble (P) fractions. Immunoblots of equal amounts of soluble and insoluble fractions were probed with anti-myc antibodies. (D) BHK21 cells transfected with myc-LULL1 and LULL1-myc were labeled with anti-myc and anti-PDI. (E) Immunoblotting of lysates from BHK21 cells transfected with myc-LAP1 or myc-LULL1 digested with PNGase F or endoglycosidase H and probed with anti-myc antibody.
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fig4: LULL1 is an ER resident protein with homology to LAP1. (A) The percentage of amino acid sequence identity of predicted nucleoplasmic (LAP1), cytoplasmic (LULL1), and transmembrane and lumenal portions of human LULL1 and LAP1. (B) CLUSTAL W alignment of human LAP1 and LULL1 amino acid sequences. Asterisk indicates position with identical amino acid residues, colon indicates conserved amino acid residues, and period indicates semi-conserved amino acid residues. Predicted membrane spanning domains (determined with TMPred) are shaded and a conserved potential N-linked glycosylation site is boxed. (C) BHK21 cells transfected with myc-LULL1 were lysed in buffer with or without 1% Triton X-100 and centrifuged to separate lysates into soluble (S) and insoluble (P) fractions. Immunoblots of equal amounts of soluble and insoluble fractions were probed with anti-myc antibodies. (D) BHK21 cells transfected with myc-LULL1 and LULL1-myc were labeled with anti-myc and anti-PDI. (E) Immunoblotting of lysates from BHK21 cells transfected with myc-LAP1 or myc-LULL1 digested with PNGase F or endoglycosidase H and probed with anti-myc antibody.

Mentions: Because the LAP1 lumenal domain appears to be a torsinA-interacting motif, we searched for other proteins containing this domain by performing a BLAST search of the NCBI database. This search identified a single novel human cDNA (GenBank/EMBL/DDBJ accession no. NM_145034) encoding a protein with a lumenal domain like LAP1, which we named LULL1 (Fig. 4, A and B). The LULL1 gene encodes a protein containing a single predicted transmembrane domain and appears to have arisen from a gene duplication event because it is located adjacent to the LAP1 gene on human chromosome 1q24. cDNA clones also exist for rat and mouse forms of LULL1, and the LAP1 and LULL1 genes are also adjacent within these genomes. In contrast to the conserved lumenal domains of LAP1 and LULL1, there is significant divergence between the NH2-terminal regions of these proteins that extend outside of the secretory pathway (Fig. 4, A and B).


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)

LULL1 is an ER resident protein with homology to LAP1. (A) The percentage of amino acid sequence identity of predicted nucleoplasmic (LAP1), cytoplasmic (LULL1), and transmembrane and lumenal portions of human LULL1 and LAP1. (B) CLUSTAL W alignment of human LAP1 and LULL1 amino acid sequences. Asterisk indicates position with identical amino acid residues, colon indicates conserved amino acid residues, and period indicates semi-conserved amino acid residues. Predicted membrane spanning domains (determined with TMPred) are shaded and a conserved potential N-linked glycosylation site is boxed. (C) BHK21 cells transfected with myc-LULL1 were lysed in buffer with or without 1% Triton X-100 and centrifuged to separate lysates into soluble (S) and insoluble (P) fractions. Immunoblots of equal amounts of soluble and insoluble fractions were probed with anti-myc antibodies. (D) BHK21 cells transfected with myc-LULL1 and LULL1-myc were labeled with anti-myc and anti-PDI. (E) Immunoblotting of lysates from BHK21 cells transfected with myc-LAP1 or myc-LULL1 digested with PNGase F or endoglycosidase H and probed with anti-myc antibody.
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fig4: LULL1 is an ER resident protein with homology to LAP1. (A) The percentage of amino acid sequence identity of predicted nucleoplasmic (LAP1), cytoplasmic (LULL1), and transmembrane and lumenal portions of human LULL1 and LAP1. (B) CLUSTAL W alignment of human LAP1 and LULL1 amino acid sequences. Asterisk indicates position with identical amino acid residues, colon indicates conserved amino acid residues, and period indicates semi-conserved amino acid residues. Predicted membrane spanning domains (determined with TMPred) are shaded and a conserved potential N-linked glycosylation site is boxed. (C) BHK21 cells transfected with myc-LULL1 were lysed in buffer with or without 1% Triton X-100 and centrifuged to separate lysates into soluble (S) and insoluble (P) fractions. Immunoblots of equal amounts of soluble and insoluble fractions were probed with anti-myc antibodies. (D) BHK21 cells transfected with myc-LULL1 and LULL1-myc were labeled with anti-myc and anti-PDI. (E) Immunoblotting of lysates from BHK21 cells transfected with myc-LAP1 or myc-LULL1 digested with PNGase F or endoglycosidase H and probed with anti-myc antibody.
Mentions: Because the LAP1 lumenal domain appears to be a torsinA-interacting motif, we searched for other proteins containing this domain by performing a BLAST search of the NCBI database. This search identified a single novel human cDNA (GenBank/EMBL/DDBJ accession no. NM_145034) encoding a protein with a lumenal domain like LAP1, which we named LULL1 (Fig. 4, A and B). The LULL1 gene encodes a protein containing a single predicted transmembrane domain and appears to have arisen from a gene duplication event because it is located adjacent to the LAP1 gene on human chromosome 1q24. cDNA clones also exist for rat and mouse forms of LULL1, and the LAP1 and LULL1 genes are also adjacent within these genomes. In contrast to the conserved lumenal domains of LAP1 and LULL1, there is significant divergence between the NH2-terminal regions of these proteins that extend outside of the secretory pathway (Fig. 4, A and B).

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