| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
GCP170 is a peripheral membrane protein with a long coiled-coil domain. Recently, GCP170/golgin-160 was shown to be a caspase substrate during apoptosis. The cleavage of GCP170 occurred in the NH_2-terminal head of GCP170, is required for efficient disassembly of the Golgi complex during apoptosis. This suggests that cleavage of GCP170 may disrupt protein-protein interactions important for Golgi function. In the present study we have characterized the Golgi targeting of GCP170 as a first step toward understanding its function, demonstrating that the essential domain of GCP170 for Golgi localization is in the NH_2-terminal head domain. Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein (termed GCP16) interacting with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence microscopy showed that GCP16 was co-l
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ocalized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [3^H]palmitic acid 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 (Cyx^<69,72>→Ala) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when over-expressed, had no effect on protein transport, over-expression of the wild-type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport form the Golgi to the cell surface. Less
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