| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Autophagy, a major degradative pathway of cell constituents via lysosomal/vacuolar system, is a dynamic process accompanying continuous formation and changes of membrane structures. In order to better understand the mechanism of mammalian autophagy, we have pursued our studies on unique ubiquitin-like protein conjugation system indispensable for autophagy. There are two ubiquitin-like modifiers ; Apg12p and Apg8p. The two modifiers are activated by Apg7p, the common E1 enzyme, in ATP-dependent manner. Then, Apg12p is transferred to Apg10p, an E2 enzyme, and finally conjugated with Apg5p. Apg8p is transferred to Apg3p, another E2 enzyme, and then conjugated with phosphatidylethanolamine. The two conjugation reactions, which operate independently but concertedly, are necessary for autophagosome formation. During the course of this study, we have focused our attention to the interaction between Apg7p and other proteins. Our new findings are summarized as follows. 1) Apg7p exists in homodimer as revealed by yeast two-hybrid analysis and chemical cross-linking experiments. The deletion of its carboxyl 40 amino acids results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an acticve-site cysteine. 2) In mammalian cells, three Apg8p homologs (MAP-LC3, GABARAP, and GATE-16) exist. We showed that all the three homologs can be activated by Apg7p and subsequently transferred to Apg3p. It has been also found that lipidation form of MAP-LC3 is enriched in rat liver autophagosomal/autolysosomal membranes, indicating that at least MAP-LC3 is a definitive Apg8p homolog that can be targeted onto autophagosomes. 3) Overexpression of both Apg7p and Apg3p, which enhances lipidation of Apg8ps, promotes the formation of Apg12p-Apg5p conjugate. Thus, there are intimate crosstalks between the two protein conjugation pathways.
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