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GM 130及其受体GRASP 65在高尔基体的定位机制。(1)通过使用^S-甲硫氨酸的脉冲追踪亚细胞分级分离实验<35>，揭示了GM 130和GRASP 65在合成后不久定位于高尔基体。(2)形态学分析表明，新合成的GM 130和GRASP 65定位于高尔基体的条件下，从内质网到高尔基体的运输被抑制的突变体Sarlp的显微注射。(3)体外翻译的GM 130和GRASP 65与纯化的高尔基体膜特异性结合。这些结果表明，GM 130和GRASP 65直接定位于高尔基体，而不是初始靶向高尔基体，这表明GM 130和GRASP 65是支持高尔基体极化的候选结构蛋白。从基因组数据库中鉴定了作为GM 130和GRASP 65定位决定子的候选者的Yiplp家族跨膜蛋白的基因。其中一个家族成员蛋白定位于高尔基体，其过表达使高尔基体解体。高尔基体解体的分子机制正在研究中。在用低pH培养基处理细胞后，高尔基体也被分解。目前正在研究这种处理对GM 130和GRASP 65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. 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》。

Yoshimura, S. et al.: "Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus"Journal of Cell Science. 114. 41005-4115 (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。