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Vol. 17, Issue 5, 2101-2112, May 2006

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The Slp4-a Linker Domain Controls Exocytosis through Interaction with Munc18-1·Syntaxin-1a Complex

Takashi Tsuboi ^*, and Mitsunori Fukuda ^* 

^* Fukuda Initiative Research Unit, Riken (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan; Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan

Submitted November 15, 2005; Revised February 6, 2006; Accepted February 8, 2006
Monitoring Editor: Adam Linstedt

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1·syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl–induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1·syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.

Abbreviations used: GFP, green fluorescent protein; HA, hemagglutinin; HRP, horseradish peroxidase; mRFP, monomeric red fluorescent protein; NPY, neuropeptide Y; SHD, Slp homology domain; siRNA, small interfering RNA; Slac2, Slp homologue lacking C2 domains; Slp, synaptotagmin-like protein; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor; VAMP-2; vesicle-associated membrane protein-2; Venus, pH-insensitive yellow fluorescent protein; TIRF, total internal reflection fluorescence.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Mitsunori Fukuda (mnfukuda{at}brain.riken.go.jp).

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