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末端区域具有高尔基体定位信号（残基号373-528），其中Tyr^<521>和Tyr^<522>对于高尔基的定位至关重要。此外，发现N端保留Phe^<93>和Phe^<94>可将内质网（ER）的退出信号作用到高尔基体。 GCP60包含与N末端区域的酰基-COA结合结构域相似的特征序列（104-164）。由于据报道酰基-COA在ER-Golgi运输中起作用，因此我们检查了GCP60是否与N.Knudsen博士（丹麦）合作，GCP60是否具有酰基-COA结合活性。然而，结果表明，即使使用了最敏感的测定方法，GCP60也没有酰基-COA结合活性。2）GCP16和GCP170的表征。 GCP16被确定为与Golfin家族成员GCP170相互作用的分子。发现GCP16与高尔基膜密切相关，尽管它没有变速箱……更多的ANE域。标记实验[^3H]棕榈酸酯和突变分析表明，GCP16在Cys^<69>和Cys^<72>上被酰化，这是其与膜的紧密相关性。没有潜在酰基化位点的突变体不再定位于高尔基体，表明通过与GCP 170相互作用，依赖性是GCP16定位的先决条件。此外，还发现GCP16本身是GCP16本身是GCPase h-/N-rass的酰基转移酶特异性的亚基，并且是摄入量。另一方面，当穿孔的细胞与细胞质和ATP孵育时，高尔基体堆栈被破坏成管状结构。在此条件下，GCP170从高尔基膜迅速释放。通过添加GCP170将高尔基体的这种结构变化回收到正常状态，表明GCP170在维持高尔基体结构中的重要作用。3）P115与COG与COG复合物的相互作用，以维持高尔基体结构； P115是COPI转运vedsicles的束缚因子，其中包含一个高度同源区域（HR2），带有酵母USO1P。酵母两杂交筛选表明，P115的HR2结构域与COG-2（保守的寡头高尔基体）配合物COG-2相互作用。当P115表达被siRNA敲除时，高尔基体络合物被碎裂并破坏。野生型P115的表达恢复了受影响细胞中正常的高尔基体结构，而HR2占用的P115引起了异常的不规则高尔基结构，这表明P115与COG复合物相互作用并在维持高尔基体结构中起作用。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