Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
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Research Abstract |
COPII-coated vesicles are involved in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. COPII consists of three parts; a small GTP binding protein, Sarlp and the two protein complexes, Sec23p-Sec24p and Sec13p-Sec31p. We have previously identified a Sec23p-interacting protein, p125, which exhibits homology with phosphatidic acid-preferring phospholipase A_1 (PA-PLA_1), and p125's cognate KIAA0725p. PA-PLA_1, p125 and KIAA0725p appear to constitutea novel family of phospholipases. In this study, we charocterized p125 and KIAA0725P in detail To assess the function of KIAA0725p, we overexpressed it in cultured mammalian cells. Overexpression of KIAA0725p, like that of p125, caused dispersion of the Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins, which mediate the tethering of transport vesicles to the target membranes, and caused aggregation of the ER. The enzyme activity of KIAA0725p was measured using phospholipid liposome substrates. We found that KIAA0725p possesses phospholipase A_1 activity preferentially for phosphatidic acid and Ser-351 if KIAA0725p is required for the activity. Under the same condition, the activity of p125 was not detected. These results suggest that the cellular function of KIAA0725p is different from that of p125 To determine the exact localization of p125, monoclonal antibodies against p125 were prepared. We found that p125, as well as the COPII, is localized to ER exit sites and its localization is regulated through the GTP/GDP cycle of Sarlp. P125 in conjunction with the COPII bound to beads bearing a peptide containing a FF motif, which is known to be a signal for export from the ER. Depletion of the cis-Golgi compartment. Our results suggest that p125 is involved in the early secretory pathway through the association with COPII
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