| Co-Investigator(Kenkyū-buntansha) |
FUJIWARA Toshiyuki Fukuoka University, School of Medicine, Instructor, 医学部, 講師 (80190099)
SOHDA Miwa Fukuoka University, School of Medicine, Research Associate, 医学部, 助手 (20258528)
|
|---|
| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
|
|---|
| Research Abstract |
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
|
