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Molecular Cell Biological Studies of Autophagy

自噬的分子细胞生物学研究

基本信息

批准号：
10480202
负责人：
OHSUMI Yoshinori
金额：
$ 9.47万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

We discovered autophagy in yeast and defined the whole process., then isolated a set of autophagy-deficient mutants, and have cloned most of APG genes essential for autophagy. We are now characterizing these gene products. We found a novel protein conjugation system required for autophagy. The C-terminal Gly of Apg12 is bound to Lys residue of Apg5 via an isopeptide bond. This conjugation reaction is distinct from but quite similar to ubiquitination. Apg10 functions as an E2 conjugating enzyme by showing thioester bond between Apg12 and Apg10. In a two-hybrid screen with Apg12 as a bait, we identified Apg16. We noticed that the Apg16 is bound to preferentially to Apg12-Apg5 conjugate. Apg16 forms an homo-oligomer via the coiled coil domain. Hence, this hetero-oligomeric large protein complex may play a role in autophagic process.Apg8 is up-regulated upon starvation. Indirect immunofluorescence study showed that intracellular localization of Apg8 drastically changes. Upon starvation, Apg8 appears adjacent to the vacuole, which represents autophagosome or its intermediate and finally autophagic bodies. According to EM analysis, Apg8 is mostly enriched on the intermediate of autophagosome. Thus, Apg8 could be a useful tracer for membrane flow in autophagosome formation. Formation process of autophagosome is accompanied with multiple fusion of small precursor structures to the intermediate structure.

我们在酵母中发现了自噬并定义了整个过程，然后分离了一组自噬缺陷突变体，并克隆了大部分自噬必需的APG基因。我们现在正在表征这些基因产物。我们发现了自噬所需的新型蛋白质缀合系统。 Apg12 的 C 端 Gly 通过异肽键与 Apg5 的 Lys 残基结合。这种缀合反应与泛素化不同但非常相似。 Apg10 通过在 Apg12 和 Apg10 之间显示硫酯键而发挥 E2 结合酶的作用。在以 Apg12 作为诱饵的双杂交筛选中，我们鉴定出了 Apg16。我们注意到 Apg16 优先与 Apg12-Apg5 缀合物结合。 Apg16 通过卷曲螺旋结构域形成同源寡聚体。因此，这种异源寡聚大蛋白复合物可能在自噬过程中发挥作用。饥饿时Apg8上调。间接免疫荧光研究表明 Apg8 的细胞内定位发生了巨大变化。饥饿时，Apg8 出现在液泡附近，液泡代表自噬体或其中间体和最终的自噬体。电镜分析显示，Apg8主要富集在自噬体中间体上。因此，Apg8 可能是自噬体形成中膜流的有用示踪剂。自噬体的形成过程伴随着小的前体结构与中间结构的多重融合。