title: Tomosyn interacts with the t-SNAREs syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation.
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.
authors.affiliation: Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, 2010 Sydney, New South Wales, Australia.
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Widberg
authors.forename: Nia J
authors.initials: NJ
authors.lastname: Bryant
authors.forename: Milena
authors.initials: M
authors.lastname: Girotti
authors.forename: Shane
authors.initials: S
authors.lastname: Rea
authors.forename: David E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology*; D002454:Cell Differentiation; D003001:Cloning, Molecular; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000502:physiology; D009479:Neuropeptides / Q000502:physiology*; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D011506:Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C100493:SNAP23 protein, human; C495693:Slc2a4 protein, mouse; C107017:Snap23 protein, mouse; C497943:Stxbp3 protein, mouse; D033921:Vesicular Transport Proteins; C112618:tomosyn protein, mouse