| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
The structure of the Golgi complex is totally dependent on its rates of exchange with the other inner-membrane system. The budding and fission are involved in a Golgi-mediated vesicular transport. Also the fission of the Golgi complex leads to extensive fragmentation during mitosis. In addition to proteinous factors, the changes in membrane phospholipids should be involved in the fission. Previous reports suggested that acyl-CoA was involved in the fission of the transport vesicles in Golgi apparatus. In the present study, we propose the importance of the reverse reactions of acyltransferases other than the endophilin and BARS in membrane dynamics during the fission and fragmentation of the Golgi complex.
We found the ATP-independent acyl-CoA synthesis in Golgi membranes. When rat liver Golgi membranes were incubated with CoA in the presence of bovine serum albumin, large amounts of acyl-CoA were formed even in the absence of ATP. The acyl-CoA formation was dependent on the concentratio
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n of CoA and followed typical Michaelis-Menten equation (Km for CoA, 100 μM. The acyl-CoA formation was totally independent on ATP. Under the same conditions, free fatty acids failed to form acyl-CoA, suggesting that the formation of acyl-CoA observed here is clearly distinct from the synthesis by acyl-CoA synthetase that ligates fatty acid and CoA in the presence of ATP. Next, we analyzed the changes in lysophospholipids and phospholipids during the acyl-CoA synthesis to search the acyl donor of the reactions. Generally lysophospholipids within the cells are known to be low. However, large amounts of Iysophosphatidylinositol and lysophosphatidylcholine were generated when liver Golgi was incubated with CoA. In contrast, the contents of phosphatidylinositol and phosphatidylcholine were decreased. These results suggest that acyl donor of ATP-independent acyl-CoA synthetic pathway is membrane phospholipids. This activity was very high ; during 30 min-incubation, 30 % of phosphatidylinositol and 10 % of phosphatidylcholine were converted to acyl-CoA and corresponding lysophospholipids. Taken together, we proposed that the reverse reaction of lysophospholipid acyltransferases is involved in the ATP-independent acyl-CoA synthetic pathway. The reverse and forward reactions of lysophospholipid acyltransferases transiently produce acyl-CoA and lysophospholipids.
We considered the physiological meaning of the reverse reaction of the acyltransferases. We expressed GFP-tagging GTase, Golgi-resident protein, in HeLa cells, and visualized Golgi apparatus. The effect of CoA loading was examined using the cells by "bead-loading method". When CoA was loaded into the cells, the Golgi was dramatically changed ; CoA induced the rapid fission and fragmentation of Golgi apparatus, and majority of the protein was found in the endoplasmic reticulum in one hr of the treatment. SH residue of CoA was important because iodeacetamide-treated CoA did not affect on the Golgi apparatus. Finally, we proposed that the reverse reaction of acyltransferases changes physiochemical propeties of membranes because lysophospholopids and fatty acyl-CoA are water-soluble, and the reverse reactions are potentially high. CoA induces the reverse reaction of acyltransferases, leading to facillitate the fission of the Golgi apparaut. The reversible change between phospholipid and lysophospholipid plus acyl-CoA by the acyltransferases may be important for the common mechanism of membrane dynamics. Less
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