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Toshihisa Ohtsuka¹, Etsuko Takao-Rikitsu¹, Eiji Inoue¹, Marie Inoue¹, Masakazu Takeuchi¹, Kaho Matsubara¹, Maki Deguchi-Tawarada¹, Keiko Satoh¹, Koji Morimoto¹, Hiroyuki Nakanishi² and Yoshimi Takai²

¹ KAN Research Institute Inc., Kyoto 600-8815, Japan
² Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan

Address correspondence to Toshihisa Ohtsuka, KAN Research Institute Inc., 93 Chudoji-Awata-cho, Shimogyo-ku, Kyoto 600-8815, Japan. Tel.: 81-75-325-5118. Fax: 81-75-325-5130. E-mail: t-ohtsuka{at}kan.gr.jp; or Yoshimi Takai, Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan. Tel.: 81-6-6879-3410. Fax: 81-6-6879-3419. E-mail: ytakai{at}molbio.med.osaka-u.ac.jp

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca²⁺-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of 120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca²⁺-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Key Words: synapse; active zone; Munc13-1; bassoon; RIM

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