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J. Biol. Chem., Vol. 279, Issue 50, 52677-52684, December 10, 2004

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Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca²⁺-dependent Exocytosis in PC12 Cells^*

Mitsunori Fukuda, Eiko Kanno, Megumi Satoh, Chika Saegusa, and Akitsugu Yamamoto¶

From the Fukuda Initiative Research Unit, RIKEN (the Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan and the ^¶Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca²⁺ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca²⁺-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca²⁺ sensor for exocytosis in endocrine cells.

Received for publication, August 12, 2004 , and in revised form, September 28, 2004.

^* This work was supported in part by grants from the Ministry of Education, Culture, Sports, and Technology of Japan (15689006 and 16044248 (to M. F.)). 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. 1 and 2.

To whom correspondence should be addressed. Tel.: 81-48-462-4994; Fax: 81-48-462-4995; E-mail: mnfukuda{at}brain.riken.go.jp.

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