2001 Fiscal Year Final Research Report Summary
Molecular mechanisms for vesicular transport and Golgi structure formation
| Project/Area Number |
10215207
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas (B)
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Fukuoka University |
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Principal Investigator |
IKEHARA Yukio School of Medicine, Professor, 医学部, 教授 (70037612)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Nobuhiro Kanazawa University, Faculty of Pharmaceutical Sciences, Assoc. Prof., 薬学部(H14年10月まで金沢大学・がん研究所), 助教授(助教授) (50294955)
MISUMI Yoshio School of Medicine, Assoc. Prof., 医学部, 助教授 (10148877)
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| Project Period (FY) |
1998 – 2001
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| Keywords | ER-Golgi transport / Golgi structure formation / Golgi targeting signal / Golgi-associated proteins / Giantin / GCP170 / GCP60 / GCP16 |
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| Research Abstract |
We have examined Golgi targeting domains of giantin, golgin-84 and syntaxin-5, all of which havea membrane-anchoring domain at the COOH terminus (CMD). Mutational analysis of the proteins showed that the cytoplasmic coiled-coil domain of about 100 residues adjacent to the CMD is essential for each protein to localize to the Golgi. Using this domain of giantin as bait in the yeast two-hybrid screening system, we identified a novel protein of 60 kDa (named GCP60) interacting with giantin. GCP60 is ubiquitously expressed and is co-localized with giantin in the Golgi. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where the COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. The results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi
GCP170, a member of the golgin family, was found to have a Golgi localization signal at the amino-terminal region. Using this domain as bait in the two-hybrid system, we identified a novel protein of 16 kDa (termed GCP16) that interacted with GCP170. GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic transmembrane domain, GCP16 was tightly associated with membranes. Labeling experiments and mutational analysis revealed that GCP16 is acylated at two positions of cysteine. 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. Overexpression of GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface, suggesting its involvement in vesicular transport from the Golgi to the cell surface.
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