Tomosyn interacts with the t-SNAREs syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation

Charlotte H. Widberg, Nia J. Bryant, Milena Girotti, Shane Rea, David E. James

Research output: Contribution to journalArticle

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Abstract

The Sec1p-like/Munc18 (SM) protein Munc18a binds to the neuronal t-SNARE Syntaxin1A and inhibits SNARE complex assembly. Tomosyn, a cytosolic Syntaxin1A-binding protein, is thought to regulate the interaction between Syntaxin1A and Munc18a, thus acting as a positive regulator of SNARE assembly. In the present study we have investigated the interaction between b-Tomosyn and the adipocyte SNARE complex involving Syntaxin4/SNAP23/VAMP-2 and the SM protein Munc18c, in vitro, and the potential involvement of Tomosyn in regulating the translocation of GLUT4 containing vesicles, in vivo. Tomosyn formed a high affinity ternary complex with Syntaxin4 and SNAP23 that was competitively inhibited by VAMP-2. Using a yeast two-hybrid assay we demonstrate that the VAMP-2-like domain in Tomosyn facilitates the interaction with Syntaxin4. Overexpression of Tomosyn in 3T3-L1 adipocytes inhibited the translocation of green fluorescent protein-GLUT4 to the plasma membrane. The SM protein Munc18c was shown to interact with the Syntaxin4 monomer, Syntaxin4 containing SNARE complexes, and the Syntaxin4/Tomosyn complex. These data suggest that Tomosyn and Munc18c operate at a similar stage of the Syntaxin4 SNARE assembly cycle, which likely primes Syntaxin4 for entry into the ternary SNARE complex.

Original languageEnglish (US)
Pages (from-to)35093-35101
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number37
DOIs
StatePublished - Sep 12 2003
Externally publishedYes

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SNARE Proteins
Munc18 Proteins
Insulin
Vesicle-Associated Membrane Protein 2
Adipocytes
Two-Hybrid System Techniques
Cell membranes
Green Fluorescent Proteins
Yeast
Assays
Carrier Proteins
Monomers
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tomosyn interacts with the t-SNAREs syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation. / Widberg, Charlotte H.; Bryant, Nia J.; Girotti, Milena; Rea, Shane; James, David E.

In: Journal of Biological Chemistry, Vol. 278, No. 37, 12.09.2003, p. 35093-35101.

Research output: Contribution to journalArticle

Widberg, Charlotte H. ; Bryant, Nia J. ; Girotti, Milena ; Rea, Shane ; James, David E. / Tomosyn interacts with the t-SNAREs syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 37. pp. 35093-35101.
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