| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2004: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2002: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Research Abstract |
In order to explore the physiological functions of ALG-2, we focused on searching for its interacting proteins. Previously, we searched for novel interacting proteins of Alix, an ALG-2 binding partner, by the yeast two-hybrid method, and obtained a candidate protein named CHMP4. We demonstrated that Alix interacts with this protein by co-immunoprecipitation assay and by subcellular co-localization by immunofluorescence microscopic analysis, suggesting involvement of Alix in endosomal sorting of endocytosed proteins. This year we demonstrated that CHMP6,an binding partner of CHMP4,is myristoylated at its N-terminus and directly interacts with EAP20. Overexpressed CHMP4b-or CHMP6-GFP protein in HeLa cells exhibited a punctate distribution in the perinuclear region, and inhibited disappearance of up-taken EGF, which is probably mediated by dominant-negative effect on suppression of transport from endosomes to lysosomes. From the network of ALG-2 interacting proteins, it seems that ALG-2 i
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s involved in membrane traffic. Indeed, Alix is suggested to play roles in protein sorting by biogenesis of multivesicular bodies (MVB, endosomes containing microvesicles in its lumen). We also identified a novel Alix interacting protein, which has a structure similar to RabGAP. This protein is localized in the vicinity of plasma membrane and cell edges, and colocalized with Alix in the filopodia and lamellipodia. On the other hand, we investigated the function of Alix in the cellular slime mold Dictyostelium discoideum, which is a model eukaryote in the boundary of unicellular and multicellular organism. Alix-gene-disrupted cells did not develop into fruiting bodies under Ca^<2+>-restricted conditions. Fruiting bodies are composed of stalk and spore, where event of programmed cell death occurs to develop into stalk. Thus, abnormalities observed in the Alix-gene-disrupted cells may be caused by impairment in cell aggregation in the early stage of development, implying that Alix is involved in cell adhesion and cell movement. Less
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