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J. Biol. Chem., Vol. 282, Issue 22, 16553-16566, June 1, 2007

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Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking^*

Matthew D'Andrea-Merrins¹, Louise Chang², Alice D. Lam, Stephen A. Ernst^¶, and Edward L. Stuenkel

From the Department of Molecular and Integrative Physiology, Life Sciences Institute, and the ^¶Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109

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 4^LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4^LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4^LE 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.

Received for publication, November 22, 2006 , and in revised form, March 12, 2007.

^* This work was supported in part by National Institute of Health (NIH) Grants NS039914 and NS053978 (to E. L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

² Supported by NIH Grants DK61618 and DK60591.

¹ To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, 7744 MSII, 1153 W. Medical Center Dr., Ann Arbor, MI 48109-0622. Tel.: 734-764-3339; Fax: 734-936-8813; E-mail: mmerrins{at}umich.edu.

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