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

研究了 GM130 及其假定受体 GRASP65 对高尔基体的定位机制。 (1)通过使用35S-甲硫氨酸的脉冲追踪亚细胞分级分离实验，揭示了GM130和GRASP65在合成后不久就定位于高尔基体。 (2)形态学分析表明，在显微注射突变体Sarlp抑制内质网向高尔基体转运的情况下，新合成的GM130和GRASP65定位于高尔基体。 (3)体外翻译的GM130和GRASP65与纯化的高尔基膜特异性结合。这些结果表明 GM130 和 GRASP65 直接定位于高尔基体，而不最初靶向高尔基体，这表明 GM130 和 GRASP65 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出 Yiplp 家族跨膜蛋白基因，这些基因是 GM130 和 GRASP65 定位决定因素的候选基因。家族成员蛋白之一位于高尔基体，其过度表达会分解高尔基体。高尔基体分解的分子机制目前正在研究中。用低pH培养基处理细胞后，高尔基体也会被分解。目前正在研究这种治疗对 GM130 和 GRASP65 的影响。

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.