Identification and characterization of taxilin isoforms

doi:10.1016/j.bbrc.2004.05.073

Biochemical and Biophysical Research Communications

Volume 319, Issue 3, 2 July 2004, Pages 936-943

https://doi.org/10.1016/j.bbrc.2004.05.073 Get rights and content

Abstract

The syntaxin family is implicated in intracellular vesicle traffic. We have recently identified taxilin, a novel syntaxin-binding protein, which has a long coiled-coil region in its C-terminal half. A database search has revealed the presence of two other molecules having a long coiled-coil region homologous to that of taxilin in mammals. Then, we here attempted to isolate and characterize the two molecules. Both the two molecules stoichiometrically interacted with several syntaxin family members. Then, we renamed original taxilin α-taxilin and named the two molecules β- and γ-taxilins, respectively. β-Taxilin was a human homologue of chicken MDP77. γ-Taxilin was an uncharacterized protein and Northern blot analysis revealed that γ-taxilin was ubiquitously expressed. β- and γ-Taxilins preferentially interacted with syntaxin-1a and -4, respectively. The taxilin family members mutually interacted with the syntaxin family members. These results indicate that there is the taxilin family composed of at least three members in mammals.

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).

References (21)

S.R Pfeffer
Rab GTPases: specifying and deciphering organelle identity and function
Trends Cell Biol.
(2001)
J.M Edwardson et al.
The secretory granule protein syncollin binds to syntaxin in a Ca²⁺-sensitive manner
Cell
(1997)
S.J Scales et al.
Amisyn, a novel syntaxin-binding protein that may regulate SNARE complex assembly
J. Biol. Chem.
(2002)
M.K Bennett et al.
The syntaxin family of vesicular transport receptors
Cell
(1993)
S Nogami et al.
Interaction of taxilin with syntaxin which does not form the SNARE complex
Biochem. Biophys. Res. Commun.
(2003)
M Nakano et al.
Interaction of syntaxin with α-fodrin, a major component of the submembranous cytoskeleton
Biochem. Biophys. Res. Commun.
(2001)
M.M Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)
A Uyeda et al.
MDP77: A novel neurite-outgrowth-promoting protein predominantly expressed in chick muscles
Biochem. Biophys. Res. Commun.
(2000)
K.E Fujimori et al.
Regulatory expression of MDP77 protein in the skeletal and cardiac muscles
FEBS Lett.
(2002)
J Pevsner et al.
Specificity and regulation of a synaptic vesicle docking complex
Neuron
(1994)

There are more references available in the full text version of this article.

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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.

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Identification and characterization of taxilin isoforms☆

Abstract

Section snippets

Experimental procedures

Involvement of an extraordinarily long coiled-coil region of taxilin in its interaction with the syntaxin family in vitro

Acknowledgements

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