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

• Project/Area Number: 13780624
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Neurochemistry/Neuropharmacology
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: 福田 光則 理化学研究所, 福田独立主幹研究ユニット, 独立主幹研究員 (50311361)
• Project Period (FY): 2001 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥2,100,000 (Direct Cost: ¥2,100,000); Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
• 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。そこで本年度は三つの異なるサブグループに属するシナプトタグミン4,5,7に対する特異的な抗体を作成し、その詳細な組織分布および細胞内局在を検討した。その結果、これら三種類のシナプトタグミンは異なる組織分布を示すだけでなく、同一の細胞においても(PC12細胞)全く異なる細胞内局在を示すことが明らかとなった。すなわちシナプトタグミン4はゴルジ体に、シナプトタグミン7はトランスゴルジネットワークに、そしてシナプトタグミン5は有芯顆粒上に存在していた(Biochem. J. (2002)365,173-180;J. Biol. Chem. (2002)277, 24499-24505;J. Biol. Chem.(2003)278,in press)。これらのシナプトタグミンはいずれも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が、スペーサー領域ではアイソフォーム間の保存性が特に低くなっている。従って、シナプトタグミン4,5,7の細胞内分布の違いはスペーサー領域に起因するものと予想され、実際、欠失変異体を用いた解析からシナプトタグミン4分子においてはスペーサー領域がゴルジ体局在化には不可欠であることが明らかとなった。

Research Products

• [Publications] Fukuda, M.Kuroda, T.S., Mikoshiba, K.: "Slac2-a/melanophilin, the missing link between Rab27 and myosin Va : Implications of a tripartite protein complex for melanosome transport"J. Biol. Chem.. 277. 12432-12436 (2002)
• [Publications] Zhang, X., Kim-Miler, M.J., Fukuda, M., Kowalchyk, J.A., Martin, T.F.J.: "Ca^<2+> -dependent synaptotagmin binding to SNAP-25 is essential for Ca^<2+> -triggered exocytosis"Nueron. 34. 599-611 (2002)
• [Publications] Kuroda, T.S., Fukuda, M., Ariga, H., Mikoshiba, K.: "Synaptotagmin-like protein 5 : A novel Rab27A effector with C-terminal tandem C2 domains"Biochem. Biophys. Res. Commun.. 293. 899-906 (2002)
• [Publications] Fukuda, M., Ogata, Y., Saegusa, C., Kannno, E., Mikoshiba, K.: "Alternative splicing isoforms of synaptotagmin VII in the mouse, rat, and human"Biochem. J.. 365. 173-180 (2002)
• [Publications] Saegusa, C., Fukuda, M., Mikoshiba, K.: "Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells"J. Biol. Chem.. 277. 24499-24505 (2002)
• [Publications] Ibata, K., Hashikawa, T., Tsuboi, T., Terakawa, S., Liang, F., Mizutani, A., Fukuda, M., Mikoshiba, K.: "Non-polarized distribution of synaptotagmin IV in neurons : Evidence that synaptotagmin IV is not a synaptic vesicle protein"Neurosci. Res.. 43. 401-406 (2002)
• [Publications] Fukuda, M., Katayama, E., Mikoshiba, K.: "The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization"J. Biol. Chem.. 277. 29315-29320 (2002)
• [Publications] Fukuda, M.: "Vesicle-associated membrane protein-2/synaptobrevin binding to synaptotagmin I promotes O-glycosylation of synaptotagmin I"J. Biol. Chem.. 277. 30351-30358 (2002)
• [Publications] Fukuda, M.: "The C2A domain of synaptotagmin-like protein 3 (Slp3) is an atypical calcium-dependent phospholipid-binding machine"Biochem. J.. 366. 681-687 (2002)
• [Publications] Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y, Kuroda, T.S.: "Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells"J. Biol. Chem.. 277. 39673-39678 (2002)
• [Publications] Fukuda, M.: "Slp homology domain 1 of Slac2-a/melanophilin is a critical determinant of GTP-dependent, specific binding to Rab27A"J. Biol. Chem.. 277. 40118-40124 (2002)
• [Publications] Fukua, M., Kuroda, T.S.: "Slac2-c, a novel linker protein that interacts with Rab27, myosin Va/Vlla, and actin"J. Biol. Chem.. 277. 43096-43103 (2002)
• [Publications] Peng W., Premkumar, A., Mossner, R., Fukuda, M., Lesch, K.P., Simantov, R.: "Synaptotagmin I and IV are differentially regulated in the brain by the recreational drug 3,4-methylenedioxymethamphetamine (MDMA)"Mol. Brain Res.. 108. 94-101 (2002)
• [Publications] Fukuda, M., Kanno, E., Ogata, Y., Saegusa, C., Kim, T., Peng Loh, Y., Yamamoto, A.: "Nerve growth factor-dependent sorting of synaptotagmin IV protein to mature dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells"J. Biol. Chem.. 278(in press). (2003)
• [Publications] 福田光則, 御子柴克彦: "シリーズ・バイオサイエンスの新世紀 第12巻 感覚器官と脳内情報処理 イノシトールリン脂質代謝と脳機能"日本生化学会. 10 (2002)
• [Publications] 福田光則: "生化学,74 シナプトタグミンファミリーの分子種多様性とその機能"日本生化学会. 5 (2002)
• [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)