Proteins as functional elements of the machinery for the post-Golgi transport network

蛋白质作为高尔基体后运输网络机器的功能元件

• 批准号: 10215208
• 负责人: YOSHIMORI Tamotsu
• 金额: $ 37.12万
• 依托单位: OKAZAKI NATIONAL RESEACH INSTITUTES (2001)Okazaki National Research Institutes (1998-2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10215208/
• 关键词: Autophagy; Autophagosomes; Apg proteins; ES cells; Accumulation of abnormal proteins; Platelet; Granule release; PKCα; エンドソーム; SKD1; Beclin; Rab4; 小胞輸送; 優性阻害変異体; 輸送再構成形; 顆粒放出反応; RabGTPase; AAA型ATPase; GFP; 受容体再循環; Rab蛋白質; Aab5 overlay

1) Autophagy : We found that binding of the Apg12-Apg5 conjugate to the precursor membranes is required for formation of autophagosomes. Furthermore, we showed that the conjugate forms a large complex (about 700kDa) together with other proteins. We purified one component of the complex and determined its amino acids sequence; it is a 63kDa novel protein, which binds to Apg5 at its N-terminal region. We also demonstrate that GATE 16 and GABARAP, homologues of the autophagosome peripheral membrane protein LC3, are processed in the same way as LC3 and localize to the autophagosomal membranes. Finally, we found that the unfolded protein accumulation is accelerated in the Apg5-deficient cells, suggesting that autophagy is involved in exclusion of abnormal proteins.2) Secretion in platelet : We established a in vitro reconstitution system for assaying release of serotonin stored in the dense-core granules and von Willebrand factor stored in the α granules by using the platelet whose plasma membrane are permeabilized by Streptolysin-O. Their secretion was dependent on ATP and cytosol. We purified the cytosolic factor required for the secretion and identified it as PKCα. Since PKCα was not sufficient to reconstitute the release, we are trying to identify other factors necessary for the release.

1）自噬：我们发现APG12-APG5偶联物与前体膜的结合是自噬体形成所必需的。此外，我们表明结合物与其他蛋白质一起形成了大型复合物（约700kDa）。我们纯化了复合物的一个成分，并确定了其氨基酸序列。它是一种63KDA的新型蛋白质，在其N末端区域与APG5结合。我们还证明了Gate 16和Gabarap（自噬体外围膜蛋白LC3的同源物）以与LC3相同的方式处理，并定位于自噬体膜。 Finally, we found that the unfolded protein accumulation is accelerated in the Apg5-deficiency cells, suggesting that autophagy is involved in exclusion of abnormal proteins.2) Secretion in platelet : We established a in vitro reconstitution system for asserting release of serotonin stored in the dense-core granules and von Willebrand factor stored in the α granules by using the platelet whose plasma膜通过链霉菌素-O透化。它们的分泌取决于ATP和细胞质。我们纯化了分泌所需的胞质因子，并将其识别为PKCα。由于PKCα不足以重建发布，因此我们正在尝试确定释放所需的其他因素。

项目成果

Murayama T, et al.: "Overexpression of low density lipoprotein receptor eliminates apolipoprotein B100-containing lipoproteins from circulation and markedly prevents atherogenesisi in apolipoprotein E-deficient mice."Atherosclerosis. 153. 295-302 (2000)

Murayama T 等人：“低密度脂蛋白受体的过度表达可消除循环中含有载脂蛋白 B100 的脂蛋白，并显着防止载脂蛋白 E 缺陷小鼠的动脉粥样硬化形成。” 动脉粥样硬化。

Kihara A, et al.: "Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Goligi network"EMBO reports. (in press). (2001)

Kihara A 等人：“Beclin-磷脂酰肌醇 3-激酶复合物在跨高利吉网络中发挥作用”EMBO 报道。

Ozaki,H. et al.: "Combined treatment of TNF-a and INF-g causes redistribution of junctional adhesion molecule in human endothelial cells."J.Immunol.. 163. 553-557 (1999)

尾崎，H.

Mizushima N, et al.: "Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells"J.Cell Biol.. 152. 657-667 (2001)

Mizushima N等人：“使用Apg5缺陷型小鼠胚胎干细胞对自噬体形成的解剖”J.Cell Biol.. 152. 657-667 (2001)

Kirisako T, et al.: "The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targetting pathway"J.Cell Biol.. 151. 263-275 (2000)

Kirisako T 等人：“可逆修饰调节 Apg8/Aut7 的膜结合状态，这对自噬和细胞质到液泡靶向途径至关重要”J.Cell Biol.. 151. 263-275 (2000)