University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria.
EMBO J. 2012 Nov 14;31(22):4304-17. doi: 10.1038/emboj.2012.278. Epub 2012 Oct 12.
Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre-autophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.
自噬是一种用于胞质物质批量降解的保守过程。自噬的触发导致形成双膜结合囊泡，称为自噬体。保守的 Atg5-Atg12/Atg16 复合物对于自噬体的形成是必不可少的。在这里，我们表明酵母 Atg5-Atg12/Atg16 复合物直接结合膜。膜结合由 Atg5 介导，由 Atg12 抑制，由 Atg16 激活。在使用巨大的单层囊泡和重组蛋白的完全重组系统中，我们揭示了该复合物的所有成分都需要有效地促进 Atg8 与磷脂酰乙醇胺的缀合，并且能够在该过程中为其所有成分指定精确的功能。此外，我们报告说，在体外，Atg5-Atg12/Atg16 复合物能够在不依赖 Atg8 的情况下将膜连接在一起。此外，我们表明，Atg5 的膜结合发生在其被招募到前自噬体结构之后，但对于自噬和细胞质到液泡运输是必需的，这发生在 Atg8 缀合和囊泡闭合之前的阶段。我们的发现为 Atg5-Atg12/Atg16 复合物在自噬体形成过程中的作用机制提供了重要的见解。
