| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Transport vesicles coated with the COPII complex, which is assembled from Sar1p, Sec23p-Sec24p, and Secl3p-Sec31p, are involved in protein export from the endoplasmic reticulum(ER). We previously identified a novel Sec23p-interacting protein, p125, which is only expressed in mammals and exhibits sequence homology with phosphatidic acid-preferring phospholipase A_1 (PA-PLA_1). PA-PLA_1,p125, and KIAA0725p appear to constitute an intracellular phospholipase A_1 family. In this study, we have analyzed the localization of PA-PLA_1 and chimeric proteins comprising p125 and two other members. The function of p125 has also been examined in detail using an RNA interference approach.
Our previous study showed that p125 is principally localized in ER exit sites where COPII-coated vesicles are produced, and that KIAA0725p is localized in the Golgi. Localization of PA-PLA_1 was analyzed by immunofluorescence microscopy. The results showed that PA-PLA_1 is exclusively localized in the cytosol, differing from that of p125 or KIAA0725p. A chimeric protein composed of the N-terminal region of p125 and KIAA0725p was localized in ER exit sites, but one composed of the N-terminal region of p125 and PA-PLA_1 was in cytosol, indicating that for localization to ER exit sites, the p125-specific N-terminal region is critical, and that the putative lipase domain is interchangeable with KIAA0725p but not with PA-PLA_1. To gain an insight into the function of p125, p125 mRNA was targeted for degradation by RNAi. In p125-depleted cells, the organization of ER exit sites was affected. The structure of the cis-Golgi compartment was also substantially disturbed, whereas the medial-Golgi was not. VSV-G Protein export from the ER occurred without a significant delay in p125-depleted cells. These results suggest that p125 is a mammalian-specific component of ER exit sites and participates in the organization of this compartment.
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