ANALYSIS FOR THE REGULATORY MECHANISM OF THE VESICULAR TRANSPORT BY COILED-COIL PROTEINS

卷曲蛋白对囊泡运输的调控机制分析

• 批准号: 12680688
• 负责人: NAKAMURA Nobuhiro
• 金额: $ 2.37万
• 依托单位: KANAZAWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680688/
• 关键词: GOLGI APPARATUS; VESICULAR TRANSPORT; LOCALIZATION; MEMBRANE PROTEIN

The localization mechanisms of GM130 and its presumed receptor, GRASP65 to the Golgi apparatus were investigated. (1) By pulse-chase subcellular fractionation experiments using <35>^S-methionin, it was revealed that GM130 amd GRASP65 localize to the Golgi apparatus soon after the synthesis. (2) Morphological analysis revealed that newly synthesized GM130 and GRASP65 were localized to the Golgi apparatus under the condition the transport form the ER to the Golgi apparatus was inhibited by the microinjection of mutant Sarlp. (3) In vitro translated GM130 and GRASP65 specifically bound to the purified・Golgi membrane. These results indicated that GM130 and GRASP65 localize to the Golgi apparatus directly without initial targeting to the Golgi apparatus suggesting that GM130 and GRASP65 are the candidate structural protein that support the polalization of the Golgi apparatus. The genes for Yiplp family transmembrane proteins which are candidates for the determinant of the localization of GM130 and GRASP65 were identified from genome data base. One of the family member proteins localized at the Golgi apparatus and its over expression disassembled the Golgi apparatus. The molecular mechanism for the Golgi disassembly is now under investigation. The Golgi apparatus is also disassembled after the treatment of cells with low pH medium. The effect of this treatment for GM130 and GRASP65 are currently investigated.

研究了GM130及其假定受体GRASP65在高尔基体的定位机制(1)通过使用35^S-甲硫氨酸的脉冲追踪亚细胞分级实验，揭示了GM130和GRASP65很快定位到高尔基体。 (2)形态学分析表明，新合成的GM130和GRASP65在从高尔基体转运的条件下定位于高尔基体。突变体Sarlp的显微注射抑制了ER到高尔基体。(3)体外翻译的GM130和GRASP65特异性结合到纯化的高尔基体膜。这些结果表明GM130和GRASP65直接定位到高尔基体，而不是最初靶向高尔基体。装置表明 GM130 和 GRASP65 是支持高尔基体极化的候选结构蛋白 Yiplp 家族的基因。从基因组数据库中鉴定出作为 GM130 和 GRASP65 定位决定因素的候选跨膜蛋白，其家族成员蛋白之一定位于高尔基体，其过度表达会分解高尔基体。目前正在研究中。用低 pH 培养基处理细胞后，高尔基体也会被分解。目前正在研究这种处理对 GM130 和 GRASP65 的影响。

项目成果

Nakatsu, K, bakuma, M., Matsuo, r., Arase, H., Yamasaki, S., Nakamura, N., Saito, T. and Ohno, H.: "A Di-leucine Signal in the Ubiquitin Moiety. POSSIBLE INVOLVEMENT IN UBIQUITINATION-MED IATED END OCYTOSIS."J. Biol. Chem.. 275. 26213-26219 (2000)

Nakatsu, K、bakuma, M.、Matsuo, r.、Arase, H.、Yamasaki, S.、Nakamura, N.、Saito, T. 和 Ohno, H.：“泛素部分中的双亮氨酸信号。

Sohda, M. et al.: "Identification and characterization of a novel Golgi Protein, GCP60, that interacts with the integral membrane protein giantin"Journal of Biological Chemistry. 276. 45298-45306 (2001)

Sohda, M. 等人：“一种新型高尔基体蛋白 GCP60 的鉴定和表征，该蛋白与整合膜蛋白巨蛋白相互作用”《生物化学杂志》。

Yoshimura, S. et al.: "Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus"Journal of Cell Science. 114. 41005-41015 (2001)

Yoshimura, S. 等人：“顺式高尔基体基质蛋白直接靶向高尔基体”细胞科学杂志。

Sohda, M., Misumi, Y., Yamamoto, A., Yano, A., Nakamura, N. and Ikehara, Y.: "Identification and I characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin."J. Biol. Chem.. 276. 45298-45306 (2001)

Sohda, M.、Misumi, Y.、Yamamoto, A.、Yano, A.、Nakamura, N. 和 Ikehara, Y.：“新型高尔基体蛋白 GCP60 的鉴定和表征，该蛋白与整合膜蛋白相互作用

Sohda, M. et al.: "Identification and characterization of a novel Golgi protein, GCP6O, that interacts with the integral membrane protein giantin"Journal of Biological Chemistry. 276. 45298-45306 (2001)

Sohda, M. 等人：“一种新型高尔基体蛋白 GCP6O 的鉴定和表征，该蛋白与整合膜蛋白巨蛋白相互作用”《生物化学杂志》。