Regulation of Autophagosome Membrane Dynamics by the Atg8 Family of Proteins

宏自噬是细胞内的应激反应途径，细胞通过它包装部分 胞浆输送到溶酶体中的胞浆。这种“包装”是由从头形成的 一种名为自噬小体的新细胞器，它生长并包裹胞液物质 最终溶酶体降解。自噬小体是如何形成的，尤其是自噬小体 膜通过自身的扩张和闭合来协调胞质毒素的捕获 全神贯注学习。在货物捕获和自噬体动力学中涉及的一个因素是 泛素样蛋白，ATG8。在自噬过程中，Atg8成为共价结合的 磷脂酰乙醇胺(PE)在自噬前体膜上，并通过 自噬小体的成熟过程。 我们的初步结果表明，Atg8-PE可以直接使膜变形，这可能是 未成熟的自噬小体独特的杯状形态。此外，我们还展示了几个 驱动Atg8募集的蛋白质被设计成识别自噬小体的独特特征 包括曲率。通过将跨多个蛋白质的这些低亲和力相互作用结合在一个复合体中， 这些蛋白质将实现戏剧性的靶向选择性，只对 自噬生长。一旦货物捕获完成，自噬小体关闭，曲率- 敏感部件被释放。ATG8-PE最终也必须被回收，我们描述了如何 负责Atg8-PE释放的蛋白水解酶也对膜结构和 组成。两项重要的技术进步使我们的发现成为可能。首先，我们 已经开发了各种体外重建方法来研究Atg8-PE和其他 自噬蛋白影响膜的变形和结构。尤其是，我们现在有 重组ATG8-PE在巨型单板层囊泡上的形成，包括高度易驯化的 膜操作模型，也足够大，以支持基于荧光显微镜的 蛋白质膜组织结构的询问。第二，我们现在可以想象自噬小体 三维超分辨率的中间结构，这样我们现在就可以可视化 杯状中间体及其裂变后的最终拆分。 通过这一提议，我们希望确切地展示Atg8-PE蛋白是如何协调双重的 蛋白质相互作用支持货物与蛋白质相互作用的责任-- 形成并关闭自噬小体的膜复合体。

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This “packaging” is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane coordinates the capture of cytosolic toxins with its own expansion and closure is an area of intense study. One factor implicated in both cargo-capture and autophagosome dynamics is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the preautophagosomal membrane and remains bound through the maturation process of the autophagosome. Our preliminary results suggest that Atg8-PE can directly deform the membrane perhaps contributing to the unique cup-like morphology of the immature autophagosome. Further, we show that several proteins driving Atg8 recruitment are designed to recognize unique features of the autophagosome including curvature. By combining these low affinity interactions across multiple proteins in a complex, these proteins would achieve dramatic targeting selectivity for only the transient intermediate in the autophagosome growth. Once cargo-capture is complete and the autophagosome closes, curvature- sensitive components are released. Atg8-PE must also eventually be recycled and we describe how the proteases responsible for Atg8-PE release are also sensitive to the membrane structure and composition. Our discoveries are made possible by two important technological advances. First we have developed a variety of in vitro reconstitution approaches to study how Atg8-PE and other autophagy proteins influence membrane deformation and structure. In particular, we have now reconstituted Atg8-PE formation on Giant Unilamellar Vesicles that comprise both a highly tractable membrane manipulation model and also are large enough to support fluorescent-microscopy based interrogation of protein-membrane organization. Second, we can now image autophagosome intermediate structures at super resolution in three dimensions so that we can now visualize both the cup-like intermediate and its eventual resolution following fission. With this proposal, we expect to demonstrate exactly how Atg8-PE proteins coordinate the dual responsibilities of protein-protein interaction supporting cargo encapsulation with the protein- membrane complexes that shape and close the autophagosome.