| Project/Area Number |
17380063
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Nagoya University |
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Principal Investigator |
MAKI Masatoshi Nagoya University, Graduate School of Bioagricultural Sciences, Professor (40183610)
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| Co-Investigator(Kenkyū-buntansha) |
HITOMI Kiyotaka Nagoya University, Graduate School of Bioagricultural Sciences, Associate professor (00202276)
SHIBATA Hideki Nagoya University, Graduate School of Bioagricultural Sciences, Assistant professor (30314470)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,790,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2007: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2006: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2005: ¥5,900,000 (Direct Cost: ¥5,900,000)
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| Keywords | ALG-2 / Alix / TSG101 / Sec31A / EF-hand / PLSCR3 / ESCRT / calcium-binding protein / カルシウム / 多胞性エンドソーム / エンドサイトーシス / ユビキチン / EGF / 小胞輸送 / EGFレセプター |
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| Research Abstract |
ALG-2 is a penta-EF-hand calcium-binding protein. To explore physiological functions of ALG-2, we searched for new interacting proteins of ALG-2. We identified Sec31A, a component of COPII working in ER-Golgi transport, associates with ALG-2 in a calcium-dependent manner and it colocalized with ALG-2 in the ER exit site by immunofluorescence microscopic analysis. Surprisingly, the alternatively spliced deltaGF-122 isoform of ALG-2 does not bind Alix but retains the capacity of binding to Sec31A. We also found that phospholipid scramblase 3 (PLSCR3) possesses two ALG-2-binding sites : one (ABS1) is similar to Alix-binding site and the other (ABS2) is similar to the Sec31A-binding in that the deltaGF-122 isoform retains the binding capacity. The second ALG-2-binding site in PLSCR3 does not possess Tyr as an essential amino acid residue. Mutational analyses of ALG-2 reveal that ABS I and ABS2 may interact with different surfaces of ALG-2. We classified ALG-2-bidning proteins as two classes : Alix-type and non-Alix type. We elucidated the 3D-structures of ALG-2 in calcium-free form and Alix peptide complex form. We analyzed HD-PTP and Brox as paralogues of Alix that contain Brol domain. Both HD-PTP and Brox bind CHMP4 proteins. While HD-PTP possesses Pro-rich region that associate with ALG-2 and endophilin, Brox lacks such a domain but possesses a famesylation site at its C-terminus. Brox was found to be secreted as an exosome. During the course of ESCRT studies, we identified a novel ESCRT-III related protein and named it CHMP7. Overexpression of green fluorescent protein(GFP) fused CHMP7 suppressed release of virus-like particle (VLP) of murine leukemia virus (MuLV) gag protein, suggesting roles in endosomal sorting of cargo proteins
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