title: Munc18c interaction with syntaxin 4 monomers and SNARE complex intermediates in GLUT4 vesicle trafficking.
abstract: In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.
authors.affiliation: Department of Molecular and Integrative Physiology, Life Sciences Institute, University of Michigan, Ann Arbor 48109-0622, USA. mmerrins@umich.edu
authors.forename: Matthew
authors.initials: M
authors.lastname: D'Andrea-Merrins
authors.forename: Louise
authors.initials: L
authors.lastname: Chang
authors.forename: Alice D
authors.initials: AD
authors.lastname: Lam
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ernst
authors.forename: Edward L
authors.initials: EL
authors.lastname: Stuenkel
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D022162:Cytoplasmic Vesicles / Q000235:genetics / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017510:Protein Folding; D050682:Q-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins
chemical_list: D051275:Glucose Transporter Type 4; D046912:Multiprotein Complexes; D051938:Munc18 Proteins; D050682:Q-SNARE Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C495693:Slc2a4 protein, mouse; C107017:Snap23 protein, mouse