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J. Biol. Chem., Vol. 279, Issue 53, 55924-55936, December 31, 2004

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Fluorescence Resonance Energy Transfer Reports Properties of Syntaxin1A Interaction with Munc18-1 in Vivo^*

Jiang Liu, Stephen A. Ernst¶, Svetlana E. Gladycheva, Yue Ying F. Lee, Stephen I. Lentz||, Chi S. Ho, Quanwen Li, and Edward L. Stuenkel**

From the Departments of Molecular and Integrative Physiology and ^¶Cell and Developmental Biology and the ^||Michigan Diabetes Research and Training Center, University of Michigan, Ann Arbor, Michigan 48109

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal -helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca²⁺-regulated secretory events.

Received for publication, August 31, 2004 , and in revised form, October 13, 2004.

^* This work was supported by Grant NS39914 from the National Institutes of Health. The costs of publication of this article were de-frayed 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.

Both authors contributed equally to this work.

^** To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, 7807 Medical Sciences II Bldg., The Medical School, The University of Michigan, Ann Arbor, MI 48109-0622. Tel.: 734-763-4477; Fax: 734-936-8813; E-mail: esterm{at}umich.edu.

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