[Abstract Title]. - Society for Neuroscience

Support: NINDS NS037409 (XOB)
DC002281 (DPC)
Title: Metabolic biotinylation of torsinA allows visualization of intracellular fate
Authors: J. E. FARLEY 1 , B. P. NILAND 1 , F. C. NERY 1 , J. W. HEWETT 1 , *D. P. COREY 2 ,
X. O. BREAKEFIELD 1 , B. A. TANNOUS 1 ;
1 Dept. of Neurol., Massachusetts Gen. Hosp., Boston, MA; 2 Dept Neurobiol, Harvard Med. Sch.,
Boston, MA
Abstract: TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic
reticulum (ER) and nuclear envelope (NE). Its relative position in these contiguous
compartments is thought to be determined by interaction with transmembrane proteins specific
each of these regions. A specific deletion of a glutamic acid residue in the carboxy terminal
region of torsinA (torsinAΓE) leads to the most common cause of early onset torsion dystonia
(DYT1). Here we describe a method to visualize the intracellular location of torsinA protein
within cells by incorporating within it a 15 amino acid biotin acceptor peptide (BAP) sequence
which is biotinylated upon co-expression of the bacterial biotin ligase directed to the ER.
Endogenous, metabolic biotinylation is a specific and sensitive technique which allows protein
labelling within or on the surface of cells and is compatible with immunocytochemistry and
electron microscopy for high resolution imaging. Here we monitored the effects of insertion of
the BAP sequence in five different regions within torsinA by co-transfection of BAP-fusion
expression constructs and biotin ligase in Gli36 cells. Some of these insertions acted as
mutations, biasing the relative cellular distribution of torsinA between the NE and ER as
visualized using streptavidin-fluorophore conjugates. Fusing the BAP sequence to the Cterminus
of torsinA favored NE localization, whereas an N-terminal insertion of BAP
(immediately following the signal sequence, amino acid 21) predisposed this protein to
concentrate preferentially in the ER. Inserting the BAP sequence at other positions in the protein,
such as position 71 which lies just after the hydrophobic region of the protein shows the typical
relatively even ER and NE distribution. These studies support the usefulness of BAP sequences
in determining the cellular localization of torsinA, as well as the use of BAP insertion sites as a
means of regional domain analysis of this and other proteins.
Disclosures: J.E. Farley, None; B.P. Niland, None; F.C. Nery, None; J.W. Hewett,
None; D.P. Corey , None; X.O. Breakefield, None; B.A. Tannous, None.
Poster
248. Dystonia
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm