Identification and characterization of factors required for the Golgi structure

高尔基体结构所需因素的识别和表征

基本信息

批准号：
17570100
负责人：
IKEHARA Yukio
金额：
$ 2.24万
依托单位：
Daiichi University (2006)Fukuoka University (2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

1) Characterization of GCP60 ; GCP60 was found to have a Golgi-localization signal at the C-terminal region (residue numbers 373-528), in which Tyr^<521> and Tyr^<522> were essential for the Golgi localization. In addition, The N-terminal residues Phe^<93> and Phe^<94> were found to act the exit signal from the endoplasmic reticulum (ER) to the Golgi. GCP60 contains a characteristic sequence similar with an acyl-CoA binding domain at the N-terminal region (104-164). As the acyl-CoA is reported to play a role for the ER-Golgi transport, we examined whether GCP60 has the acyl-CoA binding activity, in collaboration with Dr. N.Knudsen (Denmark). The result, however, showed that GCP60 has no acyl-CoA binding activity, even if the most sensitive assay method was used.2) Characterization of GCP16 and GCP170 ; GCP16 was identified as a molecule interacting with GCP170, a member of the golgin family. GCP16 was found to be tightly associated with the Golgi membrane, although it has no transmembr … More ane domain. Labeling experiments [^3H]palmitate and mutational analysis demonstrated that GCP16 was acylated at Cys^<69>and Cys^<72>, accounting for its tight association with the membrane. A mutant without potential acylation sites was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16 by interacting with GCP 170. In addition, it was found that GCP16 itself is a subunit of acyltransferase specific for the GTPase H-/N-ras, that was transported to the plasma membrane only after being acylated. On the other hand, when perforated cells were incubated with cytosol and ATP, the Golgi stack was disrupted into tubular structures. Under the condition GCP170 was rapidly released from the Golgi membrane. Such a structural change of the Golgi was recovered to the normal by addition of GCP170, indicating an important role of GCP170 in maintenance of the Golgi structure.3) Interaction of p115 with the COG complex for maintenance of the Golgi structure ; p115, a tethering factor of COPI transport vedsicles, contains a highly homologous region (HR2) with the yeast Uso1p. The yeast two-hybrid screening revealed that HR2 domain of p115 interacts with Cog-2, a member of COG (Conserved Oligmeric Golgi) complex. When the p115 expression was knock-downed by siRNA, the Golgi complex was fragmented and disrupted. The expression of wild-type p115 recovered the normal Golgi structure in the affected cells, whereas the HR2-lacking p115 caused an abnormal irregular Golgi structure, suggesting that p115 interacts with COG complex and plays a role in maintenance of the Golgi structure. Less

1）GCP60的表征；发现GCP60在C端区域（残基编号373-528）具有高尔基体定位信号，其中Tyr^<521>和Tyr^<522>对于高尔基体定位是必需的。此外，N端残基Phe^<93>和Phe^<94>被发现充当内质网的退出信号(ER) 至高尔基体，其 N 端区域 (104-164) 包含与酰基辅酶 A 结合结构域相似的特征序列，据报道酰基辅酶 A 在 ER-高尔基体运输中发挥作用，我们与 N.Knudsen 博士（丹麦）合作检查了 GCP60 是否具有酰基辅酶 A 结合活性，但结果显示 GCP60 没有。酰基-CoA 结合活性，即使使用最灵敏的测定方法。2) GCP16 和 GCP170 的表征；GCP16 被鉴定为与 GCP170 相互作用的分子，GCP170 被发现与高尔金家族紧密相关。高尔基膜，虽然没有跨膜结构域，但标记实验 [^3H] 棕榈酸酯和突变分析表明 GCP16 被酰化。在Cys^<69>和Cys^<72>处，解释了其与膜的紧密结合。没有潜在酰化位点的突变体不再定位于高尔基体，表明酰化是GCP16通过相互作用定位高尔基体的先决条件。与GCP 170。此外，还发现GCP16本身是GTP酶H-/N-ras特异性的酰基转移酶亚基，被转运至另一方面，当穿孔细胞与细胞质和 ATP 一起孵育时，高尔基体堆叠被破坏成管状结构，在这种情况下，GCP170 会从高尔基体膜上快速释放。添加GCP170使高尔基体恢复正常，表明GCP170在维持高尔基体结构中发挥重要作用。 3）p115与COG复合体相互作用以维持高尔基体结构高尔基体结构；p115（COPI 转运囊泡的束缚因子）包含与酵母 Uso1p 高度同源的区域（HR2）。酵母双杂交筛选显示 p115 的 HR2 结构域与 COG 成员 Cog-2 相互作用。（保守寡聚高尔基体）复合体当 p115 表达被 siRNA 敲低时，高尔基体复合体发生片段化。野生型 p115 的表达被破坏，恢复了受影响细胞中的正常高尔基体结构，而 HR2 相关的 p115 导致了异常不规则的高尔基体结构，表明 p115 与 COG 复合体相互作用并在维持高尔基体结构中发挥作用。较少的

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Modulation of D-serine levels via ubiquitin-dependent proteasomal degradation of serine macemase

通过丝氨酸糖化酶的泛素依赖性蛋白酶体降解调节 D-丝氨酸水平

DOI：
发表时间：
2006
期刊：
Journal of Biological Chemistry 281, 29
影响因子：
0
作者：
Langer;D.;Ikehara;Y.;Takebayashi;H.;Hawkes;R.;Zimmermann;H.;M.Sohda et al.;D.Langer et al.;S.K.Mishra et al.;E.Dumin et al.
通讯作者：
E.Dumin et al.

Depletion of vesicle-tethering factor p115 causes mini-stacked Golgi fragments with delayed protein transport