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Vol. 20, Issue 23, 4962-4975, December 1, 2009

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Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells

Liping Han^*,, Tiandan Jiang^*,, Gayoung A. Han^*,, Nancy T. Malintan, Li Xie^,, Li Wang^*, Frederick W. Tse^||, Herbert Y. Gaisano^*,,, Brett M. Collins^¶, Frederic A. Meunier, and Shuzo Sugita^*,

*Division of Fundamental Neurobiology, University Health Network, Toronto, Ontario, M5T 2S8, Canada; Departments of Physiology and Medicine, Faculty of Medicine, University of Toronto, Ontario, M5S 1A8, Canada; Queensland Brain Institute and School of Biomedical Science and ^¶Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia 4072; ^||Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Submitted August 19, 2009; Revised September 22, 2009; Accepted September 25, 2009
Monitoring Editor: Keith E. Mostov

InCytes from MBC

Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.

Address correspondence to: Shuzo Sugita (ssugita{at}uhnres.utoronto.ca).