| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
We developed three dimensional immunoelectron microscopy for studying endoplasmic reticulum (ER)-Golgi transport machinery, and applied this technique as fol o s.
1. 3D observation of spermatid Golgi apparatus. In spermatids of the Golgi stage, Golgi apparatus develops and participates in the formation of acrosomes. GM130, a cis Golgi protein was detected using colloidal gold-silver enhancement technique, EM sections of 250 nm in depth were observed under an electron microscope at 125kV. Then 3D structure of the Golgi apparatus was analyzed by electron microscopic tomography. As the results, it was clearly shown that GM130 is localized on cytosolic surface of the Golgi apparatus.
2. Retention of the ER membrane protein. To study the retention mechanism of an ER membrane protein; microsomal aldehyde dehydrogenase (msALDH), we express GFP protein with retention signal of msALDH at its C-terminal in culture cells. By examining the precise localization of the chimeric protein, it was shown that this protein was excluded from transport vesicle to the Golgi apparatus, and retained in the ER.
3. A new protein participating in budding of COPII vesicles from ER. We found a new protein, p125 which binds to a COPII coat protein Sec23, and showed that p125 is localized on the exit sites of the ER and participating in the formation of transport vesicle from ER (Collaboration with Professor Mituo Tagaya, Tokoyo University of Pharmacy and life Science.)
3. Using colloidal gold-silver enhancement technique improved in this project, we studied dynamics of the Golgi matrix proteins after a block of the ER-Golgi traffic (Collaboration with Associate Professor Nobuhiro Nakamura, Kanazawa University), accumulation of type IV collagen in the ER in Hsp47 knock-out mice (Collaboration with Professor Kazuhiro Nagata, Kyoto university), and fusion of ER membrane by BNIP1 an apoptosis related protein(Collaboration with Professor Mituo Tagaya, Tokoyo University of Pharmacy and life Science.)
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