Characterization of human torsinA and its dystonia-associated mutant form.
Deletion of a single glutamate in torsinA correlates with early-onset dystonia, the most severe form of a neurological disorder characterized by uncontrollable muscle contractions. TorsinA is targeted to the ER (endoplasmic reticulum) in eukaryotic cells. We investigated the processing and membrane association of torsinA and the dystonia-associated Glu-deletion mutant (torsinAdeltaE). We found that the signal sequence of torsinA (residues 1-20 from the 40 amino-acid long N-terminal hydrophobic region) is cleaved in Drosophila S2 cells, as shown by the N-terminal sequencing after partial protein purification. TorsinA is not secreted from S2 cells. Consistently, sodium carbonate extraction and Triton X-114 treatment showed that torsinA is associated with the ER membrane in CHO (Chinese-hamster ovary) cells. In contrast, a variant of torsinA that contains the native signal sequence without the hydrophobic region Ile24-Pro40 does not associate with the membranes in CHO cells, and a truncated torsinA without the 40 N-terminal amino acids is secreted in the S2 culture. Thus the 20-amino-acid-long hydrophobic segment in torsinA, which remains at the N-terminus after signal-peptide cleavage, is responsible for the membrane anchoring of torsinA. TorsinAdeltaE showed similar cleavage of the 20 N-terminal amino acids and membrane association properties similar to wild-type torsinA but, unlike the wild-type, torsinAdeltaE was not secreted in the S2 culture even after deletion of the membrane-anchoring segment. This indicates that the dystonia-associated mutation produces a structurally distinct, possibly misfolded, form of torsinA, which cannot be properly processed in the secretory pathway of eukaryotic cells.
Liu
Zhonghua
Z
Department of Biochemistry, Kansas State University, 104 Willard Hall, Manhattan, KS 66506, USA.
Zolkiewska
Anna
A
Zolkiewski
Michal
M
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D004331:Drosophila melanogaster; D004422:Dystonia Musculorum Deformans / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D002352:Carrier Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011994:Recombinant Proteins; C108175:TOR1A protein, human