2001 Fiscal Year Annual Research Report

シナプトタグミンファミリーの膜局在化シグナルの解析

Research Project

Project/Area Number	13780624
Research Category	Grant-in-Aid for Encouragement of Young Scientists (A)
Research Institution	The Institute of Physical and Chemical Research
Principal Investigator	福田光則理化学研究所, 発生神経生物研究チーム, 研究員 (50311361)
Keywords	シナプトタグミン / 小胞輸送 / 神経突起伸長 / C2ドメイン / エキソサイトーシス / 神経伝達物質放出 / イノシトールポリリン酸 / 膜局在化シグナル
Research Abstract	シナプトタグミンはマウスにおいて遺伝子レベルで少なくとも13種類のアイソフォームが存在することが知られているが、シナプトタグミン1以外のアイソフォームの機能、組織分布及び細胞内局在はほとんど明らかにされていない。申請者はこれまでの研究からシナプトタグミン1に関しては神経終末(axon)のシナプス小胞に、シナプトタグミン4に関してはゴルジ体から主にポストシナプス(dendrite)方向へ輸送される小胞(膜)に局在することを明らかにしている。そこで本年度はまずこれら二つのアイソフォームが何故異なる小胞に局在するのかを分子レベルで明らかにするため、シナプトタグミン4分子のゴルジ体局在化シグナルの同定を試みた。シナプトタグミン、ファミリーはいずれも6つのドメイン(細胞外アミノ末端ドメイン、膜貫通領域、スペーサードメイン、C2Aドメイン、C2Bドメイン、カルボキシル末端ドメイン)から構成されているので、まず各ドメインあるいは幾つかのドメインを欠損した様々な変異体タンパク質(T7及びFLGAタグ付き)を作成した。次にこれらの欠失変異体をPC12細胞(シナプトタグミン4を内在的に発現する細胞株)に強制発現させ、免疫組織・細胞膜分画、電子顕微鏡による解析を行った結果、アイソフォーム間で保存性の最も低いスペーサー領域がシナプトタグミン4分子のゴルジ体局在化には不可欠であることが明かとなった(Fukuda, M., Ibata, K.and Mikoshiba, K.(2001)J.Neurochem.77,730-740)。

Research Products
(17 results)

All Other

All Publications (17 results)

[Publications] Fukuda, M., Mikoshiba, K.: "Characterization of KIAA1427 protein as an atypical synaptotagmin(Syt XIII)"Biochem. J.. 354. 249-257 (2001)
[Publications] Fukuda, M., Mikoshiba, K.: "Synaptotagmin-like protein 1-3: A novel family of C-terminal-type tandem C2 proteins"Biochem. Biophys. Res. Commun.. 281. 1226-1233 (2001)
[Publications] Nalefski, E.A., Wisner, M.A., Chen, J.Z., Sprang, S.R., Fukuda, M., Mikoshiba, K.Falke, J.J.: "C2 domains from different Ca^<2+> signaling pathways display functional and mechanistic diversity"Biochemistry. 40. 3089-3100 (2001)
[Publications] Gut, A., Kiraly, C.E., Fukuda, M., Mikoshiba, K., Wollheim, C.B., Lang, J.: "Expression and localisation of synaptotagmin isoforms in endocrine β-cells : Their function in insulin exocytosis"J. Cell Sci.. 114. 1709-1716 (2001)
[Publications] Fukuda, M., Ibata, K., Mikoshiba, K.: "A unique spacer domain of synaptotagmin IV is essential for Golgi localization"J. Neurochem.. 77. 730-740 (2001)
[Publications] Fukuda, M., Saegusa, C., Mikoshiba, K.: "Novel splicing isoforms of synaptotagmin-like proteins 2 and 3: Identification of the Slp homology domain"Biochem. Biophys. Res. Commun.. 283. 513-519 (2001)
[Publications] Fukuda, M., Saegusa, C., Kanno, B., Mikoshiba, K.: "The C2A domain of double C2 protein γ contains a functional nuclear localization signal"J. Biol. Chem.. 276. 24441-24444 (2001)
[Publications] Fukuda, M., Mikoshiba, K.: "Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains"J. Biol. Chem.. 276. 27670-27676 (2001)
[Publications] Michaut, M., De Blas, G., Tomes, C.N., Yunes, R., Fukuda, M., Mayorga, L.S.: "Synaptotagmin VI participates in the acrosome reaction of human Spermatozoa"Dev. Biol.. 235. 521-529 (2001)
[Publications] Fukuda, M., Mikoshiba, K.: "Tac2-N, an atypical C-type tandem C2 protein localized in the nucleus"FEBS Lett.. 503. 217-218 (2001)
[Publications] Fukuda, M., Kanno, E., Ogata, Y., Mikoshiba, K.: "Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains"J. Biol. Chem.. 276. 40319-40325 (2001)
[Publications] Fukuda, M., Yamamoto, A., Mikoshiba, K.: "Formation of crystalloid endoplasmic reticulum induced by expression of synaptotagmin lacking the conserved WHXL motif in the C terminus"J. Biol. Chem.. 276. 41112-41119 (2001)
[Publications] Minagawa, T., Fukuda, M., Mikoshiba, K.: "Distinct phosphoinositide binding specificity of the Gap1 family proteins : Characterization of the pleckstrin homology domains of MRASAL and KIAA0538"Biochem. Biophys. Res. Commun.. 288. 87-90 (2001)
[Publications] Fukuda, M., Mikoshiba, K.: "The N-terminal cysteine cluster is essential for membrane targeting of B/K protein"Biochem. J.. 360. 441-448 (2001)
[Publications] Kida, Y., Sakaguchi, M., Fukuda, M., Mikoshiba, K., Mihara, K.: "Amino acid residues before the hydrophobic region which are critical for membrane translocation of the N-terminal domain of synaptotagmin II"FEBS. Lett.. 507. 341-345 (2001)
[Publications] Fukuda, M., Kowaichyk, J.A., Zhang, X., Martin, T.F.J., Mikoshiba, K.: "Synaptotagmin IX regulates Ca^<2+>-dependent secretion in PC12 cells"J. Biol. Chem.. 277(in press). (2002)
[Publications] Kuroda, T.S., Fukuda, M., Ariga, H., Mikoshiba, K.: "The Slp homology domain of synaptotagmin-like proteins 1-4 and Slac2 functions as a novel Rab27A binding domain"J. Biol. Chem.. 277(in press). (2002)

2001 Fiscal Year Annual Research Report

シナプトタグミンファミリーの膜局在化シグナルの解析

Principal Investigator

福田 光則 理化学研究所, 発生神経生物研究チーム, 研究員 (50311361)

Research Products

[Publications] Fukuda, M., Mikoshiba, K.: "Characterization of KIAA1427 protein as an atypical synaptotagmin(Syt XIII)"Biochem. J.. 354. 249-257 (2001)

[Publications] Fukuda, M., Mikoshiba, K.: "Synaptotagmin-like protein 1-3: A novel family of C-terminal-type tandem C2 proteins"Biochem. Biophys. Res. Commun.. 281. 1226-1233 (2001)

[Publications] Nalefski, E.A., Wisner, M.A., Chen, J.Z., Sprang, S.R., Fukuda, M., Mikoshiba, K.Falke, J.J.: "C2 domains from different Ca^<2+> signaling pathways display functional and mechanistic diversity"Biochemistry. 40. 3089-3100 (2001)

[Publications] Gut, A., Kiraly, C.E., Fukuda, M., Mikoshiba, K., Wollheim, C.B., Lang, J.: "Expression and localisation of synaptotagmin isoforms in endocrine β-cells : Their function in insulin exocytosis"J. Cell Sci.. 114. 1709-1716 (2001)

[Publications] Fukuda, M., Ibata, K., Mikoshiba, K.: "A unique spacer domain of synaptotagmin IV is essential for Golgi localization"J. Neurochem.. 77. 730-740 (2001)

[Publications] Fukuda, M., Saegusa, C., Mikoshiba, K.: "Novel splicing isoforms of synaptotagmin-like proteins 2 and 3: Identification of the Slp homology domain"Biochem. Biophys. Res. Commun.. 283. 513-519 (2001)

[Publications] Fukuda, M., Saegusa, C., Kanno, B., Mikoshiba, K.: "The C2A domain of double C2 protein γ contains a functional nuclear localization signal"J. Biol. Chem.. 276. 24441-24444 (2001)

[Publications] Fukuda, M., Mikoshiba, K.: "Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains"J. Biol. Chem.. 276. 27670-27676 (2001)

[Publications] Michaut, M., De Blas, G., Tomes, C.N., Yunes, R., Fukuda, M., Mayorga, L.S.: "Synaptotagmin VI participates in the acrosome reaction of human Spermatozoa"Dev. Biol.. 235. 521-529 (2001)

[Publications] Fukuda, M., Mikoshiba, K.: "Tac2-N, an atypical C-type tandem C2 protein localized in the nucleus"FEBS Lett.. 503. 217-218 (2001)

[Publications] Fukuda, M., Kanno, E., Ogata, Y., Mikoshiba, K.: "Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains"J. Biol. Chem.. 276. 40319-40325 (2001)

[Publications] Fukuda, M., Yamamoto, A., Mikoshiba, K.: "Formation of crystalloid endoplasmic reticulum induced by expression of synaptotagmin lacking the conserved WHXL motif in the C terminus"J. Biol. Chem.. 276. 41112-41119 (2001)

[Publications] Minagawa, T., Fukuda, M., Mikoshiba, K.: "Distinct phosphoinositide binding specificity of the Gap1 family proteins : Characterization of the pleckstrin homology domains of MRASAL and KIAA0538"Biochem. Biophys. Res. Commun.. 288. 87-90 (2001)

[Publications] Fukuda, M., Mikoshiba, K.: "The N-terminal cysteine cluster is essential for membrane targeting of B/K protein"Biochem. J.. 360. 441-448 (2001)

[Publications] Kida, Y., Sakaguchi, M., Fukuda, M., Mikoshiba, K., Mihara, K.: "Amino acid residues before the hydrophobic region which are critical for membrane translocation of the N-terminal domain of synaptotagmin II"FEBS. Lett.. 507. 341-345 (2001)

[Publications] Fukuda, M., Kowaichyk, J.A., Zhang, X., Martin, T.F.J., Mikoshiba, K.: "Synaptotagmin IX regulates Ca^<2+>-dependent secretion in PC12 cells"J. Biol. Chem.. 277(in press). (2002)

[Publications] Kuroda, T.S., Fukuda, M., Ariga, H., Mikoshiba, K.: "The Slp homology domain of synaptotagmin-like proteins 1-4 and Slac2 functions as a novel Rab27A binding domain"J. Biol. Chem.. 277(in press). (2002)

福田光則理化学研究所, 発生神経生物研究チーム, 研究員 (50311361)