| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment where sphingomyelin(SM) synthase exists was reconstituted within semi-intact Chinese hamster ovary (CHO) cells. When [^3H]ceramide that had been produced from [^3H]sphingosine at 15℃ in perforated cells was chased at 37℃ [^3H]ceramide-to-[^3H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i. e., kinetics, ATP-dependence, and temperature-dependence), [^3H]ceramide-to-[^3H]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a CHO cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [^3H]ceramide-to-^3H]SM conversion in perforated wild-type cells, while the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgi apparatus were merged, no longer required cytosol for conversion of [^3H]ceramide to
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[^3H]SM. These results indicated that the assay of [^3H]ceramide-to-[^3H]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide, and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. Moreover, we developed a novel inhibitor of ceramide transport. (1R,3R)-HPA-12, an analog of ceramide, inhibits ATP-dependent transport of ceramide from the ER to the site of SM synthesis without appreciable inhibition of ER-to-Golgi trafficking of glycoproteins.
During the period of this research, we also made progress in the study on serine palmitoyltransfease (SPT), the enzyme catalyzing the initial step of sphingolpid biosynthesis. SPT consists of two subunits, LCB1 and LCB2. We showed that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells. Hereditary sensory neuropathy type 1 (HSN1), a dominantly inherited degenerative disorder of the peripheral nerves, is accompanied by specific misssense mutations in the LCB1 subunit. We indicated that the HSN1-associated mutations in LCB1 confer dominant-negative effects on the SPT enzyme. Less
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