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

J Biol Chem. 2003 Sep 12;278(37):35093-101. doi: 10.1074/jbc.M304261200. Epub 2003 Jun 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Carrier Proteins / metabolism*
  • Carrier Proteins / physiology*
  • Cell Differentiation
  • Cloning, Molecular
  • Glucose Transporter Type 4
  • Insulin / pharmacology*
  • Kinetics
  • Membrane Proteins / metabolism*
  • Mice
  • Molecular Sequence Data
  • Monosaccharide Transport Proteins / metabolism*
  • Munc18 Proteins
  • Muscle Proteins*
  • Nerve Tissue Proteins / metabolism
  • Neurons / physiology
  • Neuropeptides / physiology*
  • Protein Binding
  • Protein Transport / drug effects
  • Proteins / metabolism
  • Qa-SNARE Proteins
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • R-SNARE Proteins
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / metabolism
  • Vesicular Transport Proteins*

Substances

  • Carrier Proteins
  • Glucose Transporter Type 4
  • Insulin
  • Membrane Proteins
  • Monosaccharide Transport Proteins
  • Munc18 Proteins
  • Muscle Proteins
  • Nerve Tissue Proteins
  • Neuropeptides
  • Proteins
  • Qa-SNARE Proteins
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • R-SNARE Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • SNAP23 protein, human
  • Slc2a4 protein, mouse
  • Snap23 protein, mouse
  • Stxbp3 protein, mouse
  • Vesicular Transport Proteins
  • tomosyn protein, mouse

Associated data

  • GENBANK/AF516607
  • GENBANK/AF516608