Distinct Self-oligomerization Activities of Synaptotagmin Family

doi:10.1074/jbc.M001376200

Distinct Self-oligomerization Activities of Synaptotagmin Family

UNIQUE CALCIUM-DEPENDENT OLIGOMERIZATION PROPERTIES OF SYNAPTOTAGMIN VII*

From the ^‡Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, the ^¶Department of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, and the ^‖Calciosignal Net Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation (JST), 2-28-8 Honkomagome, Bunkyo-ku, Tokyo 113-0021, Japan

Abstract

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421–31427). Synaptotagmin I (Syt I), a possible Ca²⁺sensor for neurotransmitter release, showed both Ca²⁺-dependent (via the C2 domain) and -independent (via the NH₂-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca²⁺-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca²⁺-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca²⁺-dependent self-oligomerization at the cytoplasmic domain with EC₅₀ values of about 150 μm Ca²⁺. In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca²⁺-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca²⁺-dependent manner without being tethered at the NH₂-terminal domain, whereas Ca²⁺-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH₂-terminal domain.

Footnotes

↵* This work was supported in part by grants from the Science and Technology Agency of Japan (to K. M.) and Ministry of Education, Science, and Culture of Japan Grants 11780571 and 12053274 (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: mnfukuda@brain.riken.go.jp.
Published, JBC Papers in Press, June 27, 2000, DOI 10.1074/jbc.M001376200
↵2 M. Fukuda, unpublished observations.
Abbreviations:

Syt(s)

synaptotagmin(s)

HRP

horseradish peroxidase

PAGE

polyacrylamide gel electrophoresis
- Received February 18, 2000.
- Revision received June 12, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.