Identification and characterization of proteins required for the Golgi structure

高尔基体结构所需蛋白质的鉴定和表征

基本信息

批准号：
15570167
负责人：
IKEHARA Yukio
金额：
$ 2.37万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

1) Identification of Golgi localization signals of GCP60 and its association with giantin; GCP60 was identified by the yeast two-hybrid screening technique as a molecule interacting with the golgin family giantin. GCP60 was found to have a Golgi localization signal at the C-terminal half, in which Tyr^<521> and Tyr^<522> are essential for the signal. When the two residues Phe^<93> and Phe^<94> at the N-terminal side were replaced by Ala, the newly synthesized GCP60 was retained in the endoplasmic reticulum (ER), not transported to the Golgi. The results suggest that the N-terminal Phe^<93> and Phe^<94> act as the exit signal from the ER, while the C-terminal Tyr^<521> and Tyr^<522> are required for the Golgi localization of GCP60 by interacting with giantin.2) Interaction of GCP16 with the Golgi membrane and GCP170 ; GCP16 was identified as a molecule interacting with GCP170, a member of the golgin family. GCP60 was found to be tightly associated with the Golgi membrane, although it co … More ntained no transmembrane domain. Labeling experiments with [^3H)-palmitic acid and mutational analysis demonstrated that GCP60 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 GCP170.3) Interaction of p115 with the COG complex for maintenance of the Golgi structure ; p115, a tethering factor of CON transport vesicles, contains a highly homologous region (HR2) with the yeast Usolp. The yeast two- hybrid screening demonstrated that the HR2 domain of p115 interacts with Cog-2, a member of COG (conserved oligomeric 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 Golgi, suggesting that p115 interacts with the COG complex and plays a role in maintenance of the Golgi structure.4) Involvement of GCP170 in maintenance of the Golgi structure ; When perforated semi intact cells were incubated with cytosol and ATP, the Golgi stack was disrupted with formation of tubular structures from Golgi cisternae. It was found that GCP170 was rapidly released from the Golgi membrane under the condition. Such a structural change of the Golgi was recovered to the normal by the addition of GCP170 into the culture medium, indicating an important role of GCP170 in maintenance of the Golgi structure. Less

1)GCP60的高尔基体定位信号的鉴定及其与巨人蛋白的关联；通过酵母二杂交筛选技术鉴定GCP60是与高尔金家族巨人蛋白相互作用的分子，发现GCP60在C端具有高尔基体定位信号。一半，其中 Tyr^<521> 和 Tyr^<522> 对于信号至关重要，两个残基 Phe^<93> 和N端侧的Phe^<94>被Ala取代，新合成的GCP60保留在内质网(ER)中，没有转运至高尔基体。结果表明N端Phe^<93>和Phe^<94> 充当 ER 的出口信号，而 C 端 Tyr^<521> 和 Tyr^<522> 是高尔基体定位所必需的GCP60 通过与 Giantin 相互作用。2) GCP16 与高尔基体膜和 GCP170 的相互作用；GCP16 被鉴定为与 GCP170 相互作用的分子，GCP170 被发现与高尔基体膜紧密相关。 … More 不含有跨膜结构域。[^3H)-棕榈酸标记实验和突变分析表明 GCP60 是在Cys^<69>和Cys^<72>处酰化，解释其与膜的紧密结合。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定位高尔基体的先决条件。与 GCP170 相互作用。3) p115 与 COG 复合体相互作用，维持高尔基体结构；p115 是 CON 运输的束缚因子；酵母双杂交筛选表明，当 p115 表达被敲除时，p115 的 HR2 结构域与 COG（保守寡聚高尔基体）复合体成员 Cog-2 相互作用。 - 被 siRNA 抑制后，高尔基体复合体被破碎和破坏。野生型 p115 的表达恢复了受影响细胞中的正常高尔基体结构。缺乏p115的HR2导致高尔基体异常，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。4) GCP170参与维持高尔基体结构；当穿孔的半完整细胞与胞质和ATP一起孵育时，高尔基体堆叠被破坏，高尔基池形成管状结构。发现GCP170从高尔基体膜下快速释放。通过向培养基中添加GCP170，高尔基体的这种结构变化得以恢复正常，表明GCP170在维持高尔基体结构中发挥着重要作用。

项目成果

期刊论文数量（60）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dynamics of Golgi matrix proteins after a block of ER to Golgi transport

内质网到高尔基体运输受阻后高尔基体基质蛋白的动态

DOI：
发表时间：
2004
期刊：
J Biochem. 135
影响因子：
0
作者：
Yoshimura S;Yamamoto A;Misumi Y;Sohda M;Barr FA;Fujii G;Shakoori A;Ohno H;Mihara K;Nakamura N.
通讯作者：
Nakamura N.

T.Fujiwara et al.: "Direct interaction of the Golgi membrane with the endoplasmic reticulum membren caused by nordihydroguaiaretic acid"Biochemical and Biophysical Research Communication. 301,4. 927-933 (2003)

T.Fujiwara等人：“去甲二氢愈创木酸引起的高尔基膜与内质网膜的直接相互作用”生物化学和生物物理研究通讯。

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

The Handbook of Proteolytic Enzymes (2^<nd> Edition) (分担) Chapter 593. Dipeptidyl-peptidases A and B(A.J.Barrett, N.D.Rawrings and J.F.Woessner, eds.)

蛋白水解酶手册（第 2 版）（分区）第 593 章。二肽基肽酶 A 和 B（A.J.Barrett、N.D.Rawrings 和 J.F.Woessner，编辑）

DOI：
发表时间：
2004
期刊：
影响因子：
0
作者：
Y.Misumi;Y.Ikehara
通讯作者：
Y.Ikehara

Inhibitory effect of α1-antitrypsin Pittsburgh-type mutant on proinsulin processing in regulated secretary pathway of...

α1-抗胰蛋白酶匹兹堡型突变体对调节分泌途径中胰岛素原加工的抑制作用

DOI：
发表时间：
2003
期刊：
Endocrine Journal 50, 1
影响因子：
0
作者：
Yoshimura;S.;Yamamoto;A.;Misumi;Y.;Sohda;M.;Barr;F.A.;Fujii;G.;Shakoori;A.;Ohno;H.;Mihara;K.;Nakamura;Y.;S.Yashimura et al.;E.Ohta et al.;A.Ito et al.;T.Fujiwara et al.;K.Fukushima et al.;M.S.Nadal et al.;K.Ohkubo et al.
通讯作者：
K.Ohkubo et al.

Structural formation and regulation of the Golgi complex (Review)(in Japnanese)

高尔基复合体的结构形成和调节（综述）（日语）