Identification and characterization of taxilin isoforms☆
Section snippets
Experimental procedures
Materials and chemicals. GST-fusion proteins were purified from Escherichia coli according to the manufacturer’s instructions [14]. His6-tagged α-taxilin was prepared as described previously [13]. An anti-α-taxilin antibody was prepared as described previously [12]. pcDNA-Myc-taxilin (full-length), -taxilinΔC (1–206 aa), and -taxilinΔN (192–546 aa), and pGEX-syntaxin-3ΔC (1–263 aa) and -syntaxin-4ΔC (1–273 aa) were constructed as described previously [12]. pGEX-syntaxin-1aΔC (4–266 aa),
Involvement of an extraordinarily long coiled-coil region of taxilin in its interaction with the syntaxin family in vitro
Taxilin has an extraordinarily long coiled-coil region in its C-terminal half. Since a coiled-coil region is thought to be involved in a protein–protein interaction, it is possible that the extraordinarily long coiled-coil region is involved in the interaction of taxilin with the syntaxin family and/or unidentified taxilin-binding proteins. Then, we examined by use of a pull-down assay whether the extraordinarily long coiled-coil region is involved in the interaction of taxilin with the
Acknowledgements
We are grateful to Dr. R.H. Scheller for donating plasmids and to T. Namatame, H. Hirata, and H. Kaneko for technical assistance. We wish to thank Laboratory Animal Research Center and Laboratory of Analytical Instruments, Dokkyo University School of Medicine, for the use of their facilities. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (2003, 2004).
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Abbreviations: SNARE, soluble N-ethyl maleimide-sensitive factor attachment protein receptor; VAMP, vesicle-associated membrane protein; SNAP-25, synaptosomal-associated protein of 25kDa; GST, glutathione S-transferase; aa, amino acid; PCR, polymerase chain reaction; Sf, Spodoptera frugiperda; APMSF, p-amidinophenylmethansulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.