Worm Breeder's Gazette 17(3): 37 (November 1, 2003)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | KAN Research Institute Inc., 93 Chudoji-Awata-cho, Shimogyo-ku, Kyoto 600-8815, Japan |
| 2 | E-mail: t-ohtsuka@kan.gr.jp |
| 3 | Department of Molecular Biology, Graduate School of Science, Nagoya University and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya 464-8602, Japan |
| 4 | Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan |
The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. Recently, we have identified a novel CAZ protein of ~120 kDa from rat brain and named it CAST (CAZ-associated structural protein) (1). CAST has no transmembrane segment, but has four coiled-coil domains and a C-terminal consensus motif for binding to PDZ domains (2). CAST is localized at the CAZ of conventional synapses of mouse brain. CAST binds directly RIM1 and indirectly Munc13-1 presumably through RIM1, forming a ternary complex. Our cell biological analyses in primary cultured rat hippocampal neurons demonstrate that CAST plays a role at least partly in the localization of RIM1 at the CAZ.
In C. elegans, UNC-10 has been identified and characterized as an orthologue of RIM1 (3). In the mutant animals, vesicle priming was impaired, but the organization of the active zone was intact. With GenBank database search, we then identified a putative orthologue of CAST (CeCAST) (F42A6.9: GenBank accession no. AF038613) in C.elegans. CeCAST consists of 836 amino acids (aa) with several coiled-coil regions. CeCAST shows a relatively low homology to CAST over the entire sequence (~20% aa identity), but the C-terminal consensus motif of CAST was also conserved in CeCAST. Because this motif was essential for CAST binding to RIM1 in the mammalian system (1), we examined whether CeCAST binds RIM/UNC10. We constructed two CeCAST mutants, pCMV-Myc-CeCAST-1 (residues 581-836) and pCMV-Myc-CeCAST-1deltaC (residues 581-833). The extract of HEK293 cells expressing each mutant was incubated with glutathione Sepharose beads containing a GST fusion protein of RIM/UNC-10 PDZ domain. Myc-CeCAST-1 bound to the beads, but Myc-CeCAST-1deltaC did not. This result suggests that CeCAST directly binds RIM/UNC-10 and that the CAST and RIM/UNC-10 system is also conserved in C. elegans. Cell biological and genetic analyses of CeCAST are currently under way.
References
1. Ohtsuka, T., E. Takao-Rikitsu, E. Inoue, M. Inoue, M. Takeuchi, K. Matsubara, M. Deguchi-Tawarada, K. Satoh, K. Morimoto, H. Nakanishi, and Y. Takai. 2002. CAST: A Novel Protein of the Cytomatrix at the Active Zone of Synapses That Forms a Ternary Complex with RIM1 and Munc13-1. J. Cell Biol. 158: 577-590.
2. Songyang, Z., A.S. Fanning, C. Fu, J. Xu, S.M. Marfatia, A.H. Chishti, A. Crompton, A.C. Chan, J.M. Anderson, and L.C. Cantley. 1997. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science. 275:73-77.
3. Koushika S.P., J.E. Richmond, G. Hadwiger, R.M. Weimer, E.M. Jorgensen, M.L. Nonet. 2001. A post-docking role for active zone protein Rim. Nat. Neurosci. 4:997-1005.