Autophagy-related Protein 32 Acts as Autophagic Degron and Directly Initiates Mitophagy
Autophagy-related Protein 32 Acts as Autophagic Degron and Directly Initiates Mitophagy
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DOI：
10.1074/jbc.m111.299917
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Okamoto, Koji
中科院分区：
文献类型：
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作者：
Kondo-Okamoto, Noriko;Noda, Nobuo N.;Okamoto, Koji
自噬相关的线粒体选择性降解(有丝分裂)是一个进化上保守的过程，被认为是线粒体质量和数量控制的关键。在芽殖酵母中，自噬相关蛋白32(Atg32)插入线粒体外膜，其N端和C端结构域分别暴露在胞浆和线粒体膜间隙中，在有丝分裂吞噬中起重要作用。ATG32与ATG8和ATG11相互作用，ATG8是一种定位于自噬小体的泛素样蛋白，而ATG11是选择性自噬相关途径所需的支架蛋白，尽管这些相互作用的意义仍不清楚。此外，Atg32是否是启动自噬小体形成的唯一必需和充分的蛋白质还没有被解决。在这里，我们证明了Atg32 IMS结构域对于有丝分裂是必不可少的。值得注意的是，当锚定在过氧化物体上时，Atg32胞浆结构域促进了自噬依赖的过氧化物体降解，这表明Atg32含有一个与其他细胞器自噬相容的模块。X射线结晶学研究表明，Atg32与Atg8家族相互作用的基序多肽以保守的方式与Atg8结合。这个结合界面上的突变削弱了Atg32与游离形式的Atg8和有丝分裂的联系。此外，Atg32变异体不能稳定地与Atg11相互作用，在线粒体降解方面存在严重缺陷。最后，我们证明了Atg32与Atg8和Atg11在隔离膜形成和随后的自噬小体形成之前形成了一个复合体，并且独立于隔离膜的形成和随后的自噬体形成。综上所述，我们的数据表明Atg32是一个双向平台，将Atg8和Atg11招募到线粒体表面，并形成对有丝分裂至关重要的启动子复合体。
Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its Nand C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.